http://2008.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Liblint&year=&month=2008.igem.org - User contributions [en]2024-03-28T12:01:37ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Jamboree/Schedule/Practice_sessionsJamboree/Schedule/Practice sessions2008-10-30T04:04:12Z<p>Liblint: </p>
<hr />
<div>== Friday November 7 : Practice Talks sign-up sheet ==<br />
<br />
<br />
Use this sign-up sheet to sign up for a slot on Friday night (November 7) to practice your talk. Note that there will NOT be any A/V (audio/visual) support on staff. All classrooms will be unlocked and you should use them and leave them as you found them. <br />
<br />
There are a limited number of time slots available so please only choose one slot. We cannot match the room that you will ultimately give your presentation in with the practice room. This should, however, give you a chance to practice your talk in a new environment.<br />
<br />
Also, there will also be pre-registration available beginning at 6pm. Conference services will be on-site to pass out team registration boxes (see the [[Jamboree/Compete#Team_boxes | Jamboree compete]] page). <br />
<br />
<br />
(Pizza and refreshments will be available on a first-come first-serve basis)<br />
<br />
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<table class="calendar"><h2 class="date"><a name="Friday Practice">Friday, November 7</a></h2><br />
<thead><br />
<tr><br />
<th width="15%">Time</th><br />
<th>room 123</th><br />
<th>room 124</th><br />
<th>room 141</th><br />
<th>room 144</th><br />
<th>room 155</th><br />
<th>room G449</th><br />
<th>room D463</th><br />
<th>room 261*</th><br />
<th>room 262*</th><br />
<th>room 346*</th><br />
<th>room 397*</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr class="even"><br />
<th>6:00p - 6:30p</th><br />
<td>KULeuven</td><br />
<td>UC Berkeley Tools</td><br />
<td>University of Sheffield</td><br />
<td>Melbourne</td><br />
<td>Imperial College</td><br />
<td>F1</td><br />
<td>G1</td><br />
<td>H1</td><br />
<td>I1</td><br />
<td>J1</td><br />
<td>K1</td><br />
</tr><br />
<tr class="odd"><br />
<th>6:30p - 7:00p</th><br />
<td>Heidelberg</td><br />
<td>HKUSTers</td><br />
<td>IIT madras</td><br />
<td>Slovenia</td><br />
<td>E2</td><br />
<td>F2</td><br />
<td>G2</td><br />
<td>H2</td><br />
<td>I2</td><br />
<td>J2</td><br />
<td>K2</td><br />
</tr><br />
<tr class="even"><br />
<th>7:00p - 7:30p</th><br />
<td>Warsaw</td><br />
<td>UVA</td><br />
<td>UChicago</td><br />
<td>Bologna</td><br />
<td>NYMU-Taipei</td><br />
<td>F3</td><br />
<td>UNIPV-Pavia</td><br />
<td>H3</td><br />
<td>I3</td><br />
<td>J3</td><br />
<td>K3</td><br />
</tr><br />
<tr class="even"><br />
<th>7:30p - 8:00p</th><br />
<td>UCSF</td><br />
<td>Peking</td><br />
<td>Delft UT</td><br />
<td>Calgary WetWare</td><br />
<td>iHKU</td><br />
<td>F4</td><br />
<td>Valencia</td><br />
<td>H4</td><br />
<td>I4</td><br />
<td>J4</td><br />
<td>K4</td><br />
</tr><br />
<tr class="odd"><br />
<th>8:00p - 8:30p</th><br />
<td>Caltech</td><br />
<td>Tsinghua</td><br />
<td>CPU-NanJing</td><br />
<td>Paris</td><br />
<td>Washington</td><br />
<td>F5</td><br />
<td>G5</td><br />
<td>H5</td><br />
<td>I5</td><br />
<td>J5</td><br />
<td>K5</td><br />
</tr><br />
<tr class="even"><br />
<th>8:30p - 9:00p</th><br />
<td>Kyoto</td><br />
<td>Alberta_NINT</td><br />
<td>ULeth</td><br />
<td>UAlberta</td><br />
<td>USTC</td><br />
<td>F6</td><br />
<td>G6</td><br />
<td>H6</td><br />
<td>I6</td><br />
<td>J6</td><br />
<td>K6</td><br />
</tr><br />
<tr class="odd"><br />
<th>9:00p - 9:30p</th><br />
<td>Tianjin</td><br />
<td>Waterloo</td><br />
<td>Lethbridge_CCS</td><br />
<td>Rice University</td><br />
<td>Calgary_Ethics</td><br />
<td>H7</td><br />
<td>Chiba</td><br />
<td>H7</td><br />
<td>I7</td><br />
<td>J7</td><br />
<td>K7</td><br />
</tr><br />
<tr class="even"><br />
<th>9:30p - 10:00p</th><br />
<td>Utah State</td><br />
<td>Michigan</td><br />
<td>C8</td><br />
<td>D8</td><br />
<td>E8</td><br />
<td>F8</td><br />
<td>G8</td><br />
<td>H8</td><br />
<td>I8</td><br />
<td>J8</td><br />
<td>K8</td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br />
<br />
Note that rooms marked with an asterisk (*) are smaller conference rooms throughout the Stata Center. Saturday sessions will not be held in these rooms but in order to accommodate all teams who would like to practice their presentations in the 4-hour period on Friday night, we must open these rooms for practice sessions.</div>Liblinthttp://2008.igem.org/Utah_State/29_October_2008Utah State/29 October 20082008-10-30T03:58:53Z<p>Liblint: New page: Sent completed BioBricks to iGEM</p>
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<div>Sent completed BioBricks to iGEM</div>Liblinthttp://2008.igem.org/Team:Utah_State/PartsTeam:Utah State/Parts2008-10-30T03:58:15Z<p>Liblint: /* BIOBRICKS IN PROGRESS */</p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#99CCFF">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
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==BIOBRICKS==<br />
Nine BioBricks were planned as extracts from the PHB operon. Five of these were from the PHB promoter and four were from the CAB gene cassette. <br />
<br />
[[Image:PartsUSU.jpg|800px|center]]<br />
<br />
==SUBMITTED BIOBRICKS==<br />
<br />
Three PHB promoter BioBricks were successfully ligated into the pSB1A3 plasmid. These BioBricks were constructed to be tested as promoters for green fluorescent protein to determine their autonomy and effectiveness. These BioBricks are labeled in the BioBrick registry as BBa_K089004, BBa_K089005, and BBa_K089006.<br />
<br />
<br />
[[Image:USUBiobrick1.jpg|320px]][[Image:USUBiobrick2.jpg|320px]][[Image:USUBiobrick3.jpg|320px]]<br />
<br />
Miniprepped plasmid DNA from each of the BioBricks were placed into PCR tubes and labeled with their designated BioBrick number. These were placed in a 50-ml free-standing centrifuge tube and sent to iGEM for further testing and placement in the registry. <br />
<br />
[[Image:Submitted_BioBricks_2.JPG|320px]][[Image:Submitted_BioBricks_USU.JPG|320px]][[Image:Trent_Holding_BioBricks.JPG|320px]]<br />
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==BIOBRICKS IN PROGRESS==<br />
<br />
The two remaining PHB promoter regions have been successfully amplified using PCR and are in process of ligations and transformations.<br />
<br />
[[Image:USUBiobrick4.jpg|450px]][[Image:USUBiobrick5.jpg|450px]]<br />
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Two of the CAB cassette genes, phaA and phaB, have been successfully amplified using PCR and are in process of ligation and transformation. The phaC and phaCAB sequences are in earlier stages. <br />
<br />
[[Image:BiobrickPHA.jpg|440px]]<br />
[[Image:BiobrickPHAB.jpg|440px]]<br />
[[Image:BiobrickPHAC.jpg|430px]]<br />
[[Image:BiobrickPHCAB.jpg|430px]]</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:56:52Z<p>Liblint: /* BioBrick Part Assembly */</p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State|<font color="#ffffff">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#99CCFF">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
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<!--- The Mission, Experiments ---><br />
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Polyhydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
'''1. '''3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
'''2. '''Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
'''3. '''3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The ''E. coli'' strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent ''E. coli'' cells were used for all transformations, excluding one transformation using a toxin-resistant strain.<br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent ''E. coli'' cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:56:40Z<p>Liblint: /* Transformations */</p>
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== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Polyhydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
'''1. '''3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
'''2. '''Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
'''3. '''3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The ''E. coli'' strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent ''E. coli'' cells were used for all transformations, excluding one transformation using a toxin-resistant strain.<br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:56:28Z<p>Liblint: /* Organisms */</p>
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Polyhydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
'''1. '''3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
'''2. '''Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
'''3. '''3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The ''E. coli'' strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:56:10Z<p>Liblint: </p>
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== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Polyhydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
'''1. '''3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
'''2. '''Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
'''3. '''3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:55:37Z<p>Liblint: /* PHB Metabolic Pathways */</p>
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<!--- The Mission, Experiments ---><br />
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Polyhydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
'''1. '''3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
'''2. '''Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
'''3. '''3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:55:05Z<p>Liblint: /* Introduction */</p>
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<!--- The Mission, Experiments ---><br />
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Polyhydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:54:52Z<p>Liblint: /* Abstract */</p>
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<!--- The Mission, Experiments ---><br />
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== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant ''E. coli'' and in ''C. necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/TeamTeam:Utah State/Team2008-10-30T03:54:18Z<p>Liblint: /* Logan and USU */</p>
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== '''iGEM 2008 at USU''' ==<br />
[[Image:USU_iGEM.jpg|500px|right]]<br />
|'''TEAM USU BACKGROUND'''<br><br />
Utah State University is proud to be involved in the 2008 iGEM competition for its first year. The 2008 USU iGEM team consists of 4 graduate, 5 undergraduate, and 2 high school students under the supervision of faculty with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Though many project topics were seriously discussed, the team chose to study a method of monitoring polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB operon. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction.<br />
<br />
== '''Team Members''' ==<br />
{|border = "10"<br />
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'''FACULTY ADVISORS:'''<br />
*'''Scott Hinton''': Dean of the College of Engineering, USU<br />
*'''Dr. Charles Miller''': Department of Biological and Irrigation Engineering, USU<br />
*'''Dr. Ronald C. Sims''': Department of Biological and Irrigation Engineering, USU<br />
<br />
'''GRADUATE STUDENTS:''' <br />
*'''Junling Huo''': PhD student, Department of Biological and Irrigation Engineering, USU<br />
*'''Steven Merrigan''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Joseph Camire''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Kirsten Sims''': MS student, Department of Biological and Irrigation Engineering, USU <br />
<br />
'''UNDERGRADUATE STUDENTS:'''<br />
*'''Trent Mortensen''': Department of Biological and Irrigation Engineering, USU <br />
*'''Elisabeth Linton''': Department of Biological and Irrigation Engineering, USU <br />
*'''Daniel Nelson''': Department of Biological and Irrigation Engineering, USU <br />
*'''Rachel Porter''': Department of Biological and Irrigation Engineering, USU <br />
<br />
'''HIGH SCHOOL STUDENTS:'''<br />
*'''Garrett Hinton''': Sky View High School<br />
*'''Matthew Sims''': Logan High School<br />
|<br />
<gallery><br />
Image:Scott_Hinton.jpg|Dean Scott Hinton<br />
Image:Charles-Miller.jpg|Dr. Charlie Miller<br />
Image:Ron_sims.jpg|Dr. Ron Sims<br />
Image:JH.jpg|Junlng Huo<br />
Image:JC.jpg|Joseph Camire<br />
Image:Stephen_Merrigan.JPG|Stephen Merrigan<br />
Image:Libby_linton.jpg|Elisabeth Linton<br />
Image:DN.jpg|Daniel Nelson<br />
Image:rachel_249(2).jpg|Rachel Porter<br />
Image:TM.jpg|Trent Mortensen<br />
Image:Sims.jpg|Kirsten and Matthew Sims<br />
Image:GH.jpg|Garrett Hinton<br />
</gallery><br />
|}<br />
<br />
== '''Team Member Contributions''' ==<br />
*'''Junling Huo''' is a PhD student in Biological Engineering who served as an advisor for the iGEM team. He helped to answer student questions, and guide such experimental activities as primer design, electrophoresis, and DNA purification. His personal research is on ''Rhodobacter sphaeroides'', a photosynthetic bacterium.<br />
<br />
*'''Stephen Merrigan''' is currently working on a MS in Biological Engineering at Utah State studying biodiesel production from algae, and has a background in Microbiology. Stephen's primary role was as an advisor for the project. During the first few months of the project Stephen did background research on project topics. He also helped with lab work when necessary, including DNA purification. Stephen also advised on the wiki construction.<br />
<br />
*'''Joseph Camire''' advised on targeting the amplification of the full phaCAB cassette and phaC gene,optimizing the PCR process and testing of primer sets with and without prefix and suffix regions. His work also involved sequence determination and analysis of the phaCAB cassette contained in the source plasmid. He also guided work on the phaC gene, which included targeting at amplification and preparing the gene for site-directed mutagenesis for removal of a critical restriction enzyme site prior to ligation of a new phaCAB cassette for biobrick construction.<br />
<br />
*'''Kirsten Sims''' is a first-year graduate student at Utah State University in Biological and Irrigation Engineering. She received her Bacherlor's degree from Gonzaga University in Biology. Her research focuses on the development of cellulose-derived biofuel. As a member of the USU iGEM team, she was involved in the development and design of the project, as well as participation in the laboratory procedures. She also contributed to the development of the wiki by providing an abstract of the project.<br />
<br />
*'''Trent Mortensen''' is a finishing senior in Biological Engineering. His main research direction is biomedical in nature, using herbal antibiotics as possible alternatives for disease treatment. As part of the USU iGEM team, he played an active role in the planning of the project, group coordination, advising professor consultation, laboratory work, ordering of materials, lab maintenance, Wiki preparation, documentation, and presentation preparations throughout the course of the project.<br />
<br />
*'''Elisabeth Linton''' is a Biological Engineering student. Her research is focused on polyhydroxyalkanoates. For the iGEM team, Libbie helped in project determination and planning, as well as literature review and topic research. She also carried out work in the laboratory like restriction enzyme digestions, gel electrophoresis, DNA isolation, and bacterial transformations. She was also responsible for coordinating efforts and organizing the wiki.<br />
<br />
*'''Daniel Nelson''' is pursuing a degree in Biological Engineering and his current personal research project is “Omega-3 Fatty Acid Production and Extraction in Schizochytrium limacinum SR21.” He has enjoyed participating in group discussions as project ideas and direction have been determined. In the lab, he has worked with the team to isolate, purify, and analyze the key DNA promoters for the PHB synthesis gene. He hopes that through study of these promoter regions, the team will find a way to increase PHB production and create a general gene expression system to monitor cellular product accumulation. Outside of the lab, he has helped to design the logo for the USU team.<br />
<br />
*'''Rachel Porter''' is a sophomore at Utah State University majoring in Biological Engineering/pre-med. For the iGEM project, she helped to carry out some of the lab work. In the lab, she helped isolate and purify DNA, prepare and run electrophoresis gels, and culture cells.<br />
<br />
*'''Matthew Sims''' is a Senior attending Logan High School. This is his first year participating in laboratory research at Utah State University. He plans to go to college next fall and continue his studies in molecular biology and biochemistry. He helped during the summer months to carry out many experiments and obtain and analyze data.<br />
<br />
*'''Garrett Hinton''' is a junior at Sky View High School in Smithfield, Utah. He was heavily involved in the project discussion meetings, as well as the background literature research. Garrett spent many hours working on this project and carried out a substantial portion of the laboratory work.<br />
<br />
'''''We would like to thank our faculty advisers - Dr. Ron Sims, Dean Scott Hinton, and Dr. Charlie Miller - for all of their support and for providing this opportunity for the students.'''''<br />
[[Image:Advisors.jpg|350px|center]]<br />
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== '''Logan and USU''' ==<br />
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<b><a href="http://www.usu.edu/">Utah State University</a></b> is located in Logan, Utah. Logan is about 85 miles north of Salt Lake City, Utah. The city of Logan is located in the heart of Cache Valley near the on the western slopes of the Bear River Mountains, the northernmost branch of the Wasatch Range. The city has a population of approximately 47,000. Logan was established in 1859 and has a rich heritage and wonderful culture. The city of Logan has been stated to be among the safest cities in America.<br><br />
<br><br />
Utah State University was established in 1888 as the Agricultural College of Utah. It's name was later changed to Utah State Agricultural College and was again changed to Utah State University (USU) in 1957. As the land-grant university in Utah, USU conducts world-class research in a variety of agricultural and natural resource disciplines, and has several projects in conjunction with the Department of Defense, NASA. Utah State University also conducts extensive aerospace research. The main campus is located in Logan, Utah. Beyond the Logan campus, Utah State's Extension programs extend academic resources and support throughout the entire state of Utah, having extension locations in each of Utah's 29 counties.<br><br />
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''The Sant Building, shown below, is home to the new USU synthetic biology laboratory''<br><br />
<br />
[[Image:Sant_Blgd.jpg|600px|center]]</div>Liblinthttp://2008.igem.org/Team:Utah_State/TeamTeam:Utah State/Team2008-10-30T03:53:50Z<p>Liblint: /* Team Member Contributions */</p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State|<font color="#ffffff">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#99CCFF">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
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== '''iGEM 2008 at USU''' ==<br />
[[Image:USU_iGEM.jpg|500px|right]]<br />
|'''TEAM USU BACKGROUND'''<br><br />
Utah State University is proud to be involved in the 2008 iGEM competition for its first year. The 2008 USU iGEM team consists of 4 graduate, 5 undergraduate, and 2 high school students under the supervision of faculty with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Though many project topics were seriously discussed, the team chose to study a method of monitoring polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB operon. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction.<br />
<br />
== '''Team Members''' ==<br />
{|border = "10"<br />
|-<br />
|rowspan="3"|<br />
'''FACULTY ADVISORS:'''<br />
*'''Scott Hinton''': Dean of the College of Engineering, USU<br />
*'''Dr. Charles Miller''': Department of Biological and Irrigation Engineering, USU<br />
*'''Dr. Ronald C. Sims''': Department of Biological and Irrigation Engineering, USU<br />
<br />
'''GRADUATE STUDENTS:''' <br />
*'''Junling Huo''': PhD student, Department of Biological and Irrigation Engineering, USU<br />
*'''Steven Merrigan''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Joseph Camire''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Kirsten Sims''': MS student, Department of Biological and Irrigation Engineering, USU <br />
<br />
'''UNDERGRADUATE STUDENTS:'''<br />
*'''Trent Mortensen''': Department of Biological and Irrigation Engineering, USU <br />
*'''Elisabeth Linton''': Department of Biological and Irrigation Engineering, USU <br />
*'''Daniel Nelson''': Department of Biological and Irrigation Engineering, USU <br />
*'''Rachel Porter''': Department of Biological and Irrigation Engineering, USU <br />
<br />
'''HIGH SCHOOL STUDENTS:'''<br />
*'''Garrett Hinton''': Sky View High School<br />
*'''Matthew Sims''': Logan High School<br />
|<br />
<gallery><br />
Image:Scott_Hinton.jpg|Dean Scott Hinton<br />
Image:Charles-Miller.jpg|Dr. Charlie Miller<br />
Image:Ron_sims.jpg|Dr. Ron Sims<br />
Image:JH.jpg|Junlng Huo<br />
Image:JC.jpg|Joseph Camire<br />
Image:Stephen_Merrigan.JPG|Stephen Merrigan<br />
Image:Libby_linton.jpg|Elisabeth Linton<br />
Image:DN.jpg|Daniel Nelson<br />
Image:rachel_249(2).jpg|Rachel Porter<br />
Image:TM.jpg|Trent Mortensen<br />
Image:Sims.jpg|Kirsten and Matthew Sims<br />
Image:GH.jpg|Garrett Hinton<br />
</gallery><br />
|}<br />
<br />
== '''Team Member Contributions''' ==<br />
*'''Junling Huo''' is a PhD student in Biological Engineering who served as an advisor for the iGEM team. He helped to answer student questions, and guide such experimental activities as primer design, electrophoresis, and DNA purification. His personal research is on ''Rhodobacter sphaeroides'', a photosynthetic bacterium.<br />
<br />
*'''Stephen Merrigan''' is currently working on a MS in Biological Engineering at Utah State studying biodiesel production from algae, and has a background in Microbiology. Stephen's primary role was as an advisor for the project. During the first few months of the project Stephen did background research on project topics. He also helped with lab work when necessary, including DNA purification. Stephen also advised on the wiki construction.<br />
<br />
*'''Joseph Camire''' advised on targeting the amplification of the full phaCAB cassette and phaC gene,optimizing the PCR process and testing of primer sets with and without prefix and suffix regions. His work also involved sequence determination and analysis of the phaCAB cassette contained in the source plasmid. He also guided work on the phaC gene, which included targeting at amplification and preparing the gene for site-directed mutagenesis for removal of a critical restriction enzyme site prior to ligation of a new phaCAB cassette for biobrick construction.<br />
<br />
*'''Kirsten Sims''' is a first-year graduate student at Utah State University in Biological and Irrigation Engineering. She received her Bacherlor's degree from Gonzaga University in Biology. Her research focuses on the development of cellulose-derived biofuel. As a member of the USU iGEM team, she was involved in the development and design of the project, as well as participation in the laboratory procedures. She also contributed to the development of the wiki by providing an abstract of the project.<br />
<br />
*'''Trent Mortensen''' is a finishing senior in Biological Engineering. His main research direction is biomedical in nature, using herbal antibiotics as possible alternatives for disease treatment. As part of the USU iGEM team, he played an active role in the planning of the project, group coordination, advising professor consultation, laboratory work, ordering of materials, lab maintenance, Wiki preparation, documentation, and presentation preparations throughout the course of the project.<br />
<br />
*'''Elisabeth Linton''' is a Biological Engineering student. Her research is focused on polyhydroxyalkanoates. For the iGEM team, Libbie helped in project determination and planning, as well as literature review and topic research. She also carried out work in the laboratory like restriction enzyme digestions, gel electrophoresis, DNA isolation, and bacterial transformations. She was also responsible for coordinating efforts and organizing the wiki.<br />
<br />
*'''Daniel Nelson''' is pursuing a degree in Biological Engineering and his current personal research project is “Omega-3 Fatty Acid Production and Extraction in Schizochytrium limacinum SR21.” He has enjoyed participating in group discussions as project ideas and direction have been determined. In the lab, he has worked with the team to isolate, purify, and analyze the key DNA promoters for the PHB synthesis gene. He hopes that through study of these promoter regions, the team will find a way to increase PHB production and create a general gene expression system to monitor cellular product accumulation. Outside of the lab, he has helped to design the logo for the USU team.<br />
<br />
*'''Rachel Porter''' is a sophomore at Utah State University majoring in Biological Engineering/pre-med. For the iGEM project, she helped to carry out some of the lab work. In the lab, she helped isolate and purify DNA, prepare and run electrophoresis gels, and culture cells.<br />
<br />
*'''Matthew Sims''' is a Senior attending Logan High School. This is his first year participating in laboratory research at Utah State University. He plans to go to college next fall and continue his studies in molecular biology and biochemistry. He helped during the summer months to carry out many experiments and obtain and analyze data.<br />
<br />
*'''Garrett Hinton''' is a junior at Sky View High School in Smithfield, Utah. He was heavily involved in the project discussion meetings, as well as the background literature research. Garrett spent many hours working on this project and carried out a substantial portion of the laboratory work.<br />
<br />
'''''We would like to thank our faculty advisers - Dr. Ron Sims, Dean Scott Hinton, and Dr. Charlie Miller - for all of their support and for providing this opportunity for the students.'''''<br />
[[Image:Advisors.jpg|350px|center]]<br />
<br />
== '''Logan and USU''' ==<br />
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</style> <br />
<b><a href="http://www.usu.edu/">Utah State University</a></b> is located in Logan, Utah. Logan is about 85 miles north of Salt Lake City, Utah. The city of Logan is located in the heart of Cache Valley near the on the western slopes of the Bear River Mountains, the northernmost branch of the Wasatch Range. The city has a population of approximately 47,000. Logan was established in 1859 and has a rich heritage and wonderful culture. The city of Logan has been stated to be among the safest cities in America.<br><br />
<br><br />
Utah State University was established in 1888 as the Agricultural College of Utah. It's name was later changed to Utah State Agricultural College and was again changed to Utah State University (USU) in 1957. As the land-grant university in Utah, USU conducts world-class research in a variety of agricultural and natural resource disciplines, and has several projects in conjunction with the Department of Defense, NASA. Utah State University also conducts extensive aerospace research. The main campus is located in Logan, Utah. Beyond the Logan campus, Utah State's Extension programs extend academic resources and support throughout the entire state of Utah, having extension locations in each of Utah's 29 counties.<br><br />
<br />
<br />
</html><br />
''The Sant Building, shown below, is home to the new USU synthetic biology laboratory''<br />
[[Image:Sant_Blgd.jpg|600px|center]]</div>Liblinthttp://2008.igem.org/Team:Utah_State/TeamTeam:Utah State/Team2008-10-30T03:52:46Z<p>Liblint: /* iGEM 2008 at USU */</p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State|<font color="#ffffff">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#99CCFF">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
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{|align="justify"<br />
== '''iGEM 2008 at USU''' ==<br />
[[Image:USU_iGEM.jpg|500px|right]]<br />
|'''TEAM USU BACKGROUND'''<br><br />
Utah State University is proud to be involved in the 2008 iGEM competition for its first year. The 2008 USU iGEM team consists of 4 graduate, 5 undergraduate, and 2 high school students under the supervision of faculty with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Though many project topics were seriously discussed, the team chose to study a method of monitoring polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB operon. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction.<br />
<br />
== '''Team Members''' ==<br />
{|border = "10"<br />
|-<br />
|rowspan="3"|<br />
'''FACULTY ADVISORS:'''<br />
*'''Scott Hinton''': Dean of the College of Engineering, USU<br />
*'''Dr. Charles Miller''': Department of Biological and Irrigation Engineering, USU<br />
*'''Dr. Ronald C. Sims''': Department of Biological and Irrigation Engineering, USU<br />
<br />
'''GRADUATE STUDENTS:''' <br />
*'''Junling Huo''': PhD student, Department of Biological and Irrigation Engineering, USU<br />
*'''Steven Merrigan''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Joseph Camire''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Kirsten Sims''': MS student, Department of Biological and Irrigation Engineering, USU <br />
<br />
'''UNDERGRADUATE STUDENTS:'''<br />
*'''Trent Mortensen''': Department of Biological and Irrigation Engineering, USU <br />
*'''Elisabeth Linton''': Department of Biological and Irrigation Engineering, USU <br />
*'''Daniel Nelson''': Department of Biological and Irrigation Engineering, USU <br />
*'''Rachel Porter''': Department of Biological and Irrigation Engineering, USU <br />
<br />
'''HIGH SCHOOL STUDENTS:'''<br />
*'''Garrett Hinton''': Sky View High School<br />
*'''Matthew Sims''': Logan High School<br />
|<br />
<gallery><br />
Image:Scott_Hinton.jpg|Dean Scott Hinton<br />
Image:Charles-Miller.jpg|Dr. Charlie Miller<br />
Image:Ron_sims.jpg|Dr. Ron Sims<br />
Image:JH.jpg|Junlng Huo<br />
Image:JC.jpg|Joseph Camire<br />
Image:Stephen_Merrigan.JPG|Stephen Merrigan<br />
Image:Libby_linton.jpg|Elisabeth Linton<br />
Image:DN.jpg|Daniel Nelson<br />
Image:rachel_249(2).jpg|Rachel Porter<br />
Image:TM.jpg|Trent Mortensen<br />
Image:Sims.jpg|Kirsten and Matthew Sims<br />
Image:GH.jpg|Garrett Hinton<br />
</gallery><br />
|}<br />
<br />
== '''Team Member Contributions''' ==<br />
*'''Junling Huo''' is a PhD student in Biological Engineering who served as an advisor for the iGEM team. He helped to answer student questions, and guide such experimental activities as primer design, electrophoresis, and DNA purification. His personal research is on ''Rhodobacter sphaeroides'', a photosynthetic bacterium.<br />
<br />
*'''Stephen Merrigan''' is currently working on a MS in Biological Engineering at Utah State studying biodiesel production from algae, and has a background in Microbiology. Stephen's primary role was as an advisor for the project. During the first few months of the project Stephen did background research on project topics. He also helped with lab work when necessary, including DNA purification. Stephen also advised on the wiki construction.<br />
<br />
*'''Joseph Camire''' advised on targeting the amplification of the full phaCAB cassette and phaC gene,optimizing the PCR process and testing of primer sets with and without prefix and suffix regions. His work also involved sequence determination and analysis of the phaCAB cassette contained in the source plasmid. He also guided work on the phaC gene, which included targeting at amplification and preparing the gene for site-directed mutagenesis for removal of a critical restriction enzyme site prior to ligation of a new phaCAB cassette for biobrick construction.<br />
<br />
*'''Kirsten Sims''' is a first-year graduate student at Utah State University in Biological and Irrigation Engineering. She received her Bacherlor's degree from Gonzaga University in Biology. Her research focuses on the development of cellulose-derived biofuel. As a member of the USU iGEM team, she was involved in the development and design of the project, as well as participation in the laboratory procedures. She also contributed to the development of the wiki by providing an abstract of the project.<br />
<br />
*'''Trent Mortensen''' is a finishing senior in Biological Engineering. His main research direction is biomedical in nature, using herbal antibiotics as possible alternatives for disease treatment. As part of the USU iGEM team, he played an active role in the planning of the project, group coordination, advising professor consultation, laboratory work, ordering of materials, lab maintenance, Wiki preparation, documentation, and presentation preparations throughout the course of the project.<br />
<br />
*'''Elisabeth Linton''' is a Biological Engineering student. Her research is focused on the the analysis and verification of gas chromatography and nuclear magnetic resonance methods for the quantification of intracellular polyhydroxyalkanoates in bacterial and environmental samples. For the iGEM team, Libbie helped in project determination and planning, as well as literature review and topic research. She also carried out work in the laboratory like restriction enzyme digestions, gel electrophoresis, DNA isolation, and bacterial transformations. She was also responsible for coordinating efforts and organizing the wiki.<br />
<br />
*'''Daniel Nelson''' is pursuing a degree in Biological Engineering and his current personal research project is “Omega-3 Fatty Acid Production and Extraction in Schizochytrium limacinum SR21.” He has enjoyed participating in group discussions as project ideas and direction have been determined. In the lab, he has worked with the team to isolate, purify, and analyze the key DNA promoters for the PHB synthesis gene. He hopes that through study of these promoter regions, the team will find a way to increase PHB production and create a general gene expression system to monitor cellular product accumulation. Outside of the lab, he has helped to design the logo for the USU team.<br />
<br />
*'''Rachel Porter''' is a sophomore at Utah State University majoring in Biological Engineering/pre-med. For the iGEM project, she helped to carry out some of the lab work. In the lab, she helped isolate and purify DNA, prepare and run electrophoresis gels, and culture cells.<br />
<br />
*'''Matthew Sims''' is a Senior attending Logan High School. This is his first year participating in laboratory research at Utah State University. He plans to go to college next fall and continue his studies in molecular biology and biochemistry. He helped during the summer months to carry out many experiments and obtain and analyze data.<br />
<br />
*'''Garrett Hinton''' is a junior at Sky View High School in Smithfield, Utah. He was heavily involved in the project discussion meetings, as well as the background literature research. Garrett spent many hours working on this project and carried out a substantial portion of the laboratory work.<br />
<br />
'''''We would like to thank our faculty advisers - Dr. Ron Sims, Dean Scott Hinton, and Dr. Charlie Miller - for all of their support and for providing this opportunity for the students.'''''<br />
[[Image:Advisors.jpg|350px|center]]<br />
<br />
== '''Logan and USU''' ==<br />
<html><br />
<style type="text/css"><br />
<br />
</style> <br />
<b><a href="http://www.usu.edu/">Utah State University</a></b> is located in Logan, Utah. Logan is about 85 miles north of Salt Lake City, Utah. The city of Logan is located in the heart of Cache Valley near the on the western slopes of the Bear River Mountains, the northernmost branch of the Wasatch Range. The city has a population of approximately 47,000. Logan was established in 1859 and has a rich heritage and wonderful culture. The city of Logan has been stated to be among the safest cities in America.<br><br />
<br><br />
Utah State University was established in 1888 as the Agricultural College of Utah. It's name was later changed to Utah State Agricultural College and was again changed to Utah State University (USU) in 1957. As the land-grant university in Utah, USU conducts world-class research in a variety of agricultural and natural resource disciplines, and has several projects in conjunction with the Department of Defense, NASA. Utah State University also conducts extensive aerospace research. The main campus is located in Logan, Utah. Beyond the Logan campus, Utah State's Extension programs extend academic resources and support throughout the entire state of Utah, having extension locations in each of Utah's 29 counties.<br><br />
<br />
<br />
</html><br />
''The Sant Building, shown below, is home to the new USU synthetic biology laboratory''<br />
[[Image:Sant_Blgd.jpg|600px|center]]</div>Liblinthttp://2008.igem.org/Team:Utah_StateTeam:Utah State2008-10-30T03:52:17Z<p>Liblint: </p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Utah_State|<font color="#99CCFF">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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<style type="text/css"><br />
body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
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{|align="justify"<br />
[[Image:IGEMBanner.jpg|960 px]]<br />
[[Image:Igemusu.jpg|350px|left|]]<br />
<br />
Utah State University is proud to be involved for the first time in the 2008 iGEM competition. The 2008 USU iGEM team consists of graduate and undergraduate students in Biological Engineering, as well as two high school students, working under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Prior to this project, none of the undergraduate or high school students had experience with genetic engineering or synthetic biology. This project provided a great opportunity for these students to learn about synthetic biology and its potential, as well as to gain a great deal of hands on experience with laboratory methods. Through team meetings and working closely together in the lab, team members were also provided with opportunities to get to know each other and the specific project material. <br />
<br />
Though many project topics were discussed, the team chose the project for developing a method of monitoring polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction. This project was carried out from May to October of 2008.<br />
<br />
<br><br />
USU is located in Logan, Utah and is nestled in the beautiful mountains of Cache Valley. For those interested in a great education and are looking for amazing skiing and other outdoor adventures and recreation, USU is perfect!<br />
[[Image:Utah.jpg|470px|left]][[Image:CacheValley.jpg|474px|right]]<br />
<br />
<br />
<br />
|}</div>Liblinthttp://2008.igem.org/Team:Utah_StateTeam:Utah State2008-10-30T03:52:06Z<p>Liblint: </p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Utah_State|<font color="#99CCFF">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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<style type="text/css"><br />
body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
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<br />
{|align="justify"<br />
[[Image:IGEMBanner.jpg|960 px]]<br />
[[Image:Igemusu.jpg|350px|left|]]<br />
<br />
Utah State University is proud to be involved for the first time in the 2008 iGEM competition. The 2008 USU iGEM team consists of graduate and undergraduate students in Biological Engineering, as well as two high school students, working under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Prior to this project, none of the undergraduate or high school students had experience with genetic engineering or synthetic biology. This project provided a great opportunity for these students to learn about synthetic biology and its potential, as well as to gain a great deal of hands on experience with laboratory methods. Through team meetings and working closely together in the lab, team members were also provided with opportunities to get to know each other and the specific project material. <br />
<br />
Though many project topics were discussed, the team chose the project for developing a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction. This project was carried out from May to October of 2008.<br />
<br />
<br><br />
USU is located in Logan, Utah and is nestled in the beautiful mountains of Cache Valley. For those interested in a great education and are looking for amazing skiing and other outdoor adventures and recreation, USU is perfect!<br />
[[Image:Utah.jpg|470px|left]][[Image:CacheValley.jpg|474px|right]]<br />
<br />
<br />
<br />
|}</div>Liblinthttp://2008.igem.org/Team:Utah_StateTeam:Utah State2008-10-30T03:51:51Z<p>Liblint: </p>
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<div>{| align="center"<br />
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
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{|align="justify"<br />
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[[Image:Igemusu.jpg|350px|left|]]<br />
<br />
Utah State University is proud to be involved for the first time in the 2008 iGEM competition. The 2008 USU iGEM team consists of graduate and undergraduate students in Biological Engineering, as well as two high school students, working under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Prior to this project, none of the undergraduate or high school students had experience with genetic engineering or synthetic biology. This project provided a great opportunity for these students to learn about synthetic biology and its potential, as well as to gain a great deal of hands on experience with laboratory methods. Through team meetings and working closely together in the lab, team members were also provided with opportunities to get to know each other and the specific project material. <br />
<br />
Though many project topics were discussed, the team chose the project for developing a method of monitoring Poly-β-hydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction. This project was carried out from May to October of 2008.<br />
<br />
<br><br />
USU is located in Logan, Utah and is nestled in the beautiful mountains of Cache Valley. For those interested in a great education and are looking for amazing skiing and other outdoor adventures and recreation, USU is perfect!<br />
[[Image:Utah.jpg|470px|left]][[Image:CacheValley.jpg|474px|right]]<br />
<br />
<br />
<br />
|}</div>Liblinthttp://2008.igem.org/Team:Utah_StateTeam:Utah State2008-10-30T03:50:36Z<p>Liblint: </p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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{|align="justify"<br />
[[Image:IGEMBanner.jpg|960 px]]<br />
[[Image:Igemusu.jpg|350px|left|]]<br />
<br />
Utah State University is proud to be involved for the first time in the 2008 iGEM competition. The 2008 USU iGEM team consists of graduate and undergraduate students in Biological Engineering, as well as two high school students, working under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Prior to this project, none of the undergraduate or high school students had experience with genetic engineering or synthetic biology. This project provided a great opportunity for these students to learn about synthetic biology and its potential, as well as to gain a great deal of hands on experience with laboratory methods. Through team meetings and working closely together in the lab, team members were also provided with opportunities to get to know each other and the specific project material. <br />
<br />
Though many project topics were seriously discussed, the team chose the project for developing a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction. This project was carried out from May to October of 2008.<br />
<br />
<br><br />
USU is located in Logan, Utah and is nestled in the beautiful mountains of Cache Valley. For those interested in a great education and are looking for amazing skiing and other outdoor adventures and recreation, USU is perfect!<br />
[[Image:Utah.jpg|470px|left]][[Image:CacheValley.jpg|474px|right]]<br />
<br />
<br />
<br />
|}</div>Liblinthttp://2008.igem.org/Team:Utah_StateTeam:Utah State2008-10-30T03:49:57Z<p>Liblint: </p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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<br />
{|align="justify"<br />
[[Image:IGEMBanner.jpg|960 px]]<br />
[[Image:Igemusu.jpg|350px|left|]]<br />
<br />
Utah State University is proud to be involved for the first time in the 2008 iGEM competition. The 2008 USU iGEM team consists of graduate and undergraduate students in Biological Engineering, as well as two high school students, working under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Prior to this project, none of the undergraduate or high school students had experience with genetic engineering or synthetic biology. This project provided a great opportunity for these students to learn about synthetic biology and its potential, as well as to gain a great deal of hands on experience with laboratory methods. Through team meetings and working closely together in the lab, team members were also provided with opportunities to get to know each other and the specific project material. <br />
<br />
Though many project topics were seriously discussed, the team chose the project for developing a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction. This project was carried out from May to October of 2008.<br />
<br />
<br><br />
USU is located in Logan, UT and is nestled in the beautiful mountains of Cache Valley. For those interested in a great education and are looking for amazing skiing and other outdoor adventures and recreation, USU is perfect!!<br />
[[Image:Utah.jpg|470px|left]][[Image:CacheValley.jpg|474px|right]]<br />
<br />
<br />
<br />
|}</div>Liblinthttp://2008.igem.org/Team:Utah_StateTeam:Utah State2008-10-30T03:48:58Z<p>Liblint: </p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Utah_State|<font color="#99CCFF">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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<br />
{|align="justify"<br />
[[Image:IGEMBanner.jpg|960 px]]<br />
[[Image:Igemusu.jpg|350px|left|]]<br />
<br />
Utah State University is proud to be involved for the first time in the 2008 iGEM competition. The 2008 USU iGEM team consists of graduate and undergraduate students in Biological Engineering, as well as two high school students, working under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Prior to this project, none of the undergraduate or high school students had experience with genetic engineering or synthetic biology. This project provided a great opportunity for these students to learn about synthetic biology and its potential, as well as to gain a great deal of hands on experience with laboratory methods. Through team meetings and working closely together in the lab, team members were also provided with opportunities to get to know each other and the specific project material. <br />
<br />
Though many project topics were seriously discussed, the team chose the project for developing a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction. This project was carried out from May to October of 2008.<br />
<br />
<br><br />
USU is located in Logan, UT and is nestled in the beautiful mountains of Cache Valley. For those interested in a great education and are looking for amazing skiing and other outdoor adventures and recreation, USU is perfect!!<br />
[[Image:Utah.jpg|450px|left]][[Image:CacheValley.jpg|450px|right]]<br />
<br />
<br />
<br />
|}</div>Liblinthttp://2008.igem.org/File:Utah.jpgFile:Utah.jpg2008-10-30T03:48:30Z<p>Liblint: uploaded a new version of "Image:Utah.jpg"</p>
<hr />
<div></div>Liblinthttp://2008.igem.org/File:CacheValley.jpgFile:CacheValley.jpg2008-10-30T03:48:18Z<p>Liblint: uploaded a new version of "Image:CacheValley.jpg"</p>
<hr />
<div></div>Liblinthttp://2008.igem.org/Team:Utah_State/PartsTeam:Utah State/Parts2008-10-30T03:46:41Z<p>Liblint: /* SUBMITTED BIOBRICKS */</p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State|<font color="#ffffff">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#99CCFF">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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<br />
==BIOBRICKS==<br />
Nine BioBricks were planned as extracts from the PHB operon. Five of these were from the PHB promoter and four were from the CAB gene cassette. <br />
<br />
[[Image:PartsUSU.jpg|800px|center]]<br />
<br />
==SUBMITTED BIOBRICKS==<br />
<br />
Three PHB promoter BioBricks were successfully ligated into the pSB1A3 plasmid. These BioBricks were constructed to be tested as promoters for green fluorescent protein to determine their autonomy and effectiveness. These BioBricks are labeled in the BioBrick registry as BBa_K089004, BBa_K089005, and BBa_K089006.<br />
<br />
<br />
[[Image:USUBiobrick1.jpg|320px]][[Image:USUBiobrick2.jpg|320px]][[Image:USUBiobrick3.jpg|320px]]<br />
<br />
Miniprepped plasmid DNA from each of the BioBricks were placed into PCR tubes and labeled with their designated BioBrick number. These were placed in a 50-ml free-standing centrifuge tube and sent to iGEM for further testing and placement in the registry. <br />
<br />
[[Image:Submitted_BioBricks_2.JPG|320px]][[Image:Submitted_BioBricks_USU.JPG|320px]][[Image:Trent_Holding_BioBricks.JPG|320px]]<br />
<br />
==BIOBRICKS IN PROGRESS==<br />
<br />
The two remaining PHB promoter regions have been successfully amplified using PCR and are in process of ligations and transformations.<br />
<br />
[[Image:USUBiobrick4.jpg|450px]][[Image:USUBiobrick5.jpg|450px]]<br />
<br />
Two of the CAB cassette genes, phaA and phaB, have been successfully amplified using PCR and are in process of ligations and transformations. The phaC and phaCAB sequences are in earlier stages. <br />
<br />
[[Image:BiobrickPHA.jpg|440px]]<br />
[[Image:BiobrickPHAB.jpg|440px]]<br />
[[Image:BiobrickPHAC.jpg|430px]]<br />
[[Image:BiobrickPHCAB.jpg|430px]]</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProtocolsTeam:Utah State/Protocols2008-10-30T03:42:43Z<p>Liblint: /* Polymerase Chain Reaction (PCR) */</p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#99CCFF">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
<br />
<!--- The Mission, Experiments ---><br />
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<br />
=== Bacterial Transformation === <br />
<br />
[[Image:TrentLab1.jpg|300px|right]]<br />
Once the target DNA has been successfully ligated into the plasmid vector, the plasmid must be transferred into the host cell for replication and cloning. In order to do this, the bacterial cells must first be made “competent.” The term “competent” is to describe a cell state in which there exist gaps or openings in the cell wall which will allow the plasmid containing the target genes to enter into the cell. Several methods to make bacterial cells competent exist, such as the calcium chloride method and electroporation. Competent cells may also be purchased commercially. The team at USU has purchased competent cells for all experiments. The following is the method used by the USU team to insert the plasmids containing the PHB biobricks into the cells. <br />
''Trent Mortensen is shown in the right image inspecting agar plates containing transforming cells''<br />
<br />
'''Method'''<br><br />
1. Ensure the necessary antibiotic agar plates have been prepared or begin their preparation now. Four plates per transformation will be necessary (two today, then two tomorrow for streaking). Also ensure that 10 ml liquid media is made up per transformation (also for tomorrow).<br><br />
2. Clean punchout tool by dipping in 10% chlorox, deionized water, deionized water again, then 80%aq ethanol. Let dry for 2 minutes and repeat cleaning procedure between punchouts. <br><br />
3. Punch out gene of choice with a twisting motion, allowing the metal to cut the paper. Use the center part of the punchout tool to dislodge the paper into a 2.5 ml microcentrifuge tube. <br> <br />
4. Add 5 μl TE buffer, place in a 42˚C water bath, and allow plasmids in the paper to elute for 20 minutes.<br />
5. Centrifuge tube(s) at 15K RPM for 3 minutes. Remove SOC media from the -20˚C freezer and leave out to thaw.<br><br />
6. Take competent cells from the -80˚C freezer and place on an ice bath.<br><br />
7. Pipette contents of tube up and down a few times then take 2 μl of the DNA solution and add to the competent cells. Ensure the pipetting is done directly into the cell solution. Let cells incubate in the ice bath for 30 minutes. Heat water bath to 42˚C.<br><br />
8. Heat shock cells in the 42˚C water bath for 30 seconds. Remove and place back in the ice bath for 2 minutes.<br><br />
9. In the hood, add 250 μl SOC media to each tube, bringing the total cell solution to 300 μl. Incubate at 37˚C for 1 hour.<br><br />
10. Get out the antibiotic agar plates. In the hood, add 200 μl of each transformed cell solution to the appropriate antibiotic plate. Use the Bunsen burner to create a “hockey stick” out of a glass pipette tip by holding over the flame until it bends. Allow to cool. Spread cell solution uniformly over the agar plate using the “hockey stick,” then before discarding, spread residual solution on the “stick” over a second plate to get more a more sparse colony distribution. <br><br />
11. Parafilm all plates and place in 37˚C incubator 12-14 hours, or overnight if that is not possible. <br><br />
<br />
=== Streak Plates and Liquid Cultures from Transformed Colonies ===<br />
<br />
After bacterial cells have been transformed, successfully transformed cells must be selected. Because 100% of the cells do not receive the desired plasmid and target gene, it is essential to select for cells that do have the target genes. Several methods are used to accomplish this, such as incorporation of antibiotic resistance and also the lac operon. The USU team has used antibiotic resistance to select for successful transformations. To do this, an antibiotic resistance gene is also added to the plasmid vector that contains the target genes. By doing so, it is possible to know that a cell was successfully transformed based on its ability to grow on an agar plate with antibiotics added. Because the cell is able to grow, the antibiotic resistance gene must be present as well as the target gene. From the agar plates containing the antibiotics, a colony is picked off and transferred into a liquid culture for further analysis and cellular production. The following is the method used by USU to clone the DNA and select for the successful transformation of the PHB biobrick into the cells.<br />
<br />
'''Method'''<br><br />
1. Prepare two 12 ml tubes per transformation, each with 5 ml media containing the appropriate antibiotic (if felt necessary). Get out antibiotic agar plates. Inspect plates from yesterday for colonies. At least two colonies are preferred, but one will do. Select two colonies and label them. <br><br />
2. Use a pipette tip to extract half of each colony and inoculate one agar plate per colony. Using a pipette with a tip, extract the other half of each colony and inoculate one liquid media tube per colony. Label all tubes and plates and place in the 37˚C incubator until tomorrow morning. <br><br />
<br />
=== Preparation for DNA Separation ===<br />
[[Image:Microcentrifuge.JPG|300px|right]]<br />
Following successful bacterial cloning and isolation, it is important to verify that the target gene is in the cell and that the plasmid is functional. To do this, it is a common practice to sequence the DNA plasmid. To obtain enough DNA for sequencing, the bacterial clones are grown in a liquid culture. The cells are harvested by centrifugation and then prepared for DNA plasmid extraction. DNA plasmid extraction can be done several ways, and the overall purpose is to lyse the cells and separate the plasmid DNA from all other cellular proteins, DNA, and debris. The following is the method used by the USU team to isolate the plasmid DNA containing the PHB biobricks. ''An Eppendorf Microcentrifuge was used in these experiments and is shown on the right.''<br />
<br />
'''Method'''<br><br />
1. Prepare two water baths, one boiling and the other 68˚C. <br> <br />
2. Centrifuge the 12 ml tubes containing the 5 ml cultures in the large centrifuge at 8K RPM for 20 min. Discard supernatant. <br> <br />
3. Resuspend cells in 200 μl of STET buffer. <br> <br />
4. Add 10 μl (if older preparation) Lysozyme (50 mg/ml) and incubate at room temperature for 5 min. <br> <br />
5. Boil for 45 sec and centrifuge for 20 min at 13K RPM (or until pellet gets tight).<br> <br />
6. Use a pipette tip to remove the pellet.<br> <br />
7. Add 5 μl RNase A (10 mg/ml) and incubate at 68˚C for 10 minutes.<br> <br />
8. Add 10 μl of 5% CTAB and incubate at room temperature for 3 min.<br> <br />
9. Centrifuge for 5 min at 13K RPM, discard supernatant, and resuspend in 300 μl of 1.2 M NaCl using a vortex mixer.<br> <br />
10. Add 750 μl of ethanol and centrifuge for 5 min at 13K RPM.<br> <br />
11. Discard supernatant, rinse pellet (cannot see) in 80% ethanol, and let tubes dry upside down with caps open.<br><br />
<br />
=== Restriction Enzyme Digestion and Electrophoresis ===<br />
<br />
Restriction enzyme digestion is the process by which an insert DNA sequence is separated from the rest of the DNA molecule. Specific knowledge of the DNA insert is needed to determine which enzyme and conditions to use during the digestion reaction. Once the DNA sequence is known and the correct enzymes have been selected, the DNA may be digested. Listed below is the procedure used by USU to digest the cellular DNA. After enzyme digestion, electrophoresis is used to separate the plasmid from the insert. A gel is prepared and the respective reaction mixes are loaded into the gel. Using a DNA ladder, and knowing the size of the insert, the corresponding band can be seen and cut out of the gel. The insert may then be removed and isolated from the gel, thus yielding the desired DNA. The DNA from this may then be used in PCR reactions, sequencing, ligations for further experimentation, and many more. Listed below are the protocols used by the USU team to run the electrophoresis reaction. <br />
<br />
'''Method'''<br><br />
1. Resuspend DNA in 40 μl water, vortex, and do a brief centrifuge to get solution to the bottom of the tube. <br><br />
2. Add components to the digestion solution in the following order: DNA (23 μl), 10X Tango buffer (3 μl), Xba1 (2 μl), and Pst1 (2 μl). The volume and restriction enzymes can be varied, but it should be ensured that the total volume is 10X the amount of Tango buffer added. Tap tubes periodically and allow to digest while preparing electrophoresis gel. <br><br />
3. Prepare electrophoresis gel by adding 2 g agarose to 200 ml TAE (1% solution). This is best done in an Erlenmeyer flask of adequate volume as swirling will need to be done. Place in the microwave and microwave on high for 20 seconds at a time, pulling it out and swirling until solution is homogeneous again, then repeating (BE CAREFUL to watch the solution closely when swirling – it superheats and can boil over and cause severe burns). Continue until solution is seen boiling in the microwave then gently swirl again. <br><br />
4. Add 20 μl ethidium bromide to solution and swirl until dissolved evenly. <br><br />
5. Add 6 μl of 6X loading dye to each tube of digested DNA solution. <br><br />
6. Prepare the electrophoresis unit by orienting the basin sideways with rubber gaskets firmly against the side. Place desired well template in the basin. <br><br />
7. When the agarose solution is cool enough to comfortably touch the flask, pour into the basin until the solution is about ¾ of the way to the top of the well template. <br><br />
8. When the gel is solidified (should look somewhat cloudy), remove the well template and change basin orientation to have the wells closest to the negative pole (as the DNA will flow towards the positive pole). Pour 1X TAE buffer into both sides of the electrophoresis unit until it just covers the gel and fills the wells.<br><br />
9. By inserting the pipette tip below the TAE liquid and into the well, add 10 μl of DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the digested DNA solutions to the wells by adding about 2 μl less than the total volume in the tubes to prevent air bubbles in the wells.<br><br />
10. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 70 V (this can be as high as 100 V if time is an issue), and press the start button. Separation should take two to three hours. The yellow dye shows the location of the smaller nucleotide lengths and the blue dye shows the location of the larger nucleotide lengths. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. The basin can then be placed back in the electrophoresis unit for further separation if desired. Take care to not have the power supply on without the lid to the unit in place. <br><br />
11. When the desired level of separation is attained, the basin can be placed on the transilluminator for picture taking. Place the cone-shaped cover over the transilluminator and place the digital camera in the top hole for pictures.<br><br />
<br />
''The electrophoresis units and UV transilluminator used in this project are shown in the left and center images below. An electrophoresis image is given on the right.''<br />
<br />
[[Image:Electrophoresis_Units.JPG|320px]][[Image:UV_Transilluminator.JPG|320px]][[Image:Elect.jpg|320px]]<br />
<br />
=== Media Preparation ===<br />
<br />
For all experimentation involving the need for bacterial biomass and experimentation, proper media is needed to grow the cells. The following is the media composition and methods used by USU to prepare the media.<br />
<br />
1. Add 5 g yeast extract, 10 g NaCl, 10 g Bacto Tryptone, and 15 g agar (if desired) to a 2 L Erlenmeyer flask and bring the volume up to 1 L with ddH20. Mix by swirling. Cover top with foil.<br><br />
2. Autoclave for 45 minutes (liquid setting, 0 minutes drying time). It will take an additional half hour for the autoclave to finish cooling then an additional 20 to 30 minutes until the media is cool enough to pour.<br><br />
<br />
=== 1X TAE Preparation ===<br />
1. Add 40 ml 50X TAE solution to a 2 L flask and bring level up to 2 L with ddH20. <br><br />
<br />
=== Polymerase Chain Reaction (PCR) ===<br />
[[Image:Thermocycler.JPG|250px|right]]<br />
<br />
PCR is used to amplify a desired DNA sequence. The reaction is first set up by designing primers that will bind only to the desired regions of the DNA sequence. Once the primer and polymerase have been selected, the reaction parameters of time and temperature must be optimized. When the reaction works properly only the target DNA will be amplified into large quantities that may then be isolated and used for further experimentation. The following is the procedure used by USU for PCR reactions to amplify the PHB synthesis and promotion genes.<br> ''An Eppendorf Thermocycle was used in these experiments, as shown in the image on the right.''<br />
<br />
'''Method'''<br><br />
1. Obtain the following reagents from the freezer: DNA template (cells or DNA), 10X Taq buffer (+KCl, -Mg/Cl<small>2</small>), MgCl<small>2</small>, 10 mM dNTP Mix, Taq polymerase (take out of freezer only immediately when needed and put back), and sterile distilled H<small>2</small>O. Place all reagents on ice. Also obtain PCR (either 0.2 or 0.5 ml) tubes.<br><br />
2. Add the following reagents to a tube (50 μl reaction) in the following volumes and order:<br><br />
• 32 μl sterile H<small>2</small>O,<br><br />
• 5 μl 10X buffer, <br><br />
• 2 μl dNTP Mix, <br><br />
• 6 μl MgCl<small>2</small><br><br />
• 3 μl cells/DNA, <br><br />
• 0.25 μl Taq Polymerase<br><br />
• 1 μl primer 1<br><br />
• 1 μl primer 2<br><br />
MgCl<small>2</small> volume can be varied (lower to increase specificity – just ensure total volume is 50 ul with H<small>2</small>O). If many reactions are to be constructed, a master mix can be made up to cut down on time and pipette tip usage (if this is done, ensure primers are added to the appropriate reaction, i.e. perhaps not to the master mix). Tap or vortex tubes and take to the thermocyler. Place all reagents back in the -20˚C freezer. <br><br />
3. Choose thermocycler temperatures. The Eppendorf Mastercycler will cycle between three temperatures: typical temperatures are 94˚C for denaturing, 50-60˚C for primer annealing, and 72˚C for polymerase extending. Lowering the annealing temperature decreases DNA specificity; 55˚C is a good temperature to begin if no trials have been made with the sample. <br><br />
4. Turn on thermocycler with the switch in the back of the unit and open the lid. The placement of the tubes depends on the size of the tube (0.2 or 0.5 ml) and whether or not a temperature gradient is to be used. <br><br><br />
<br />
* If no gradient will be used, tubes can be placed anywhere on the unit in the appropriately-sized hole. Select “Files” and press enter. Select “Load” and then “Standard.” If cells will be used in the reaction, include a 1-minute lysing step at the beginning (step 1); this will be followed by a 1-minute DNA denaturing step (step 2). If purified DNA will be used, set step 1 to 1 second. Set an annealing temperature for step 3. Ensure the lid temperature is 105˚C and the extending temperature is 72˚C. Press exit. If prompted to save, save by pressing enter three times. Press exit to return to the main menu. Choose “Start” on the main menu and select “Standard.” The program should begin. <br><br />
<br />
* If a gradient is to be used, temperature will vary according to column. A 20˚C range is the maximum range that can be used (+/- 10˚C). The range is made by setting a temperature for the middle column and then setting a +/- range. To see what the temperatures will be if a gradient is used, select “OPTIONS” on the main menu, then select “Gradient.” Select the size tube that is being used by pressing “Sel,” then press enter. Choose a temperature for the center column, press enter, then select a +/- range and press enter. The column number along with the corresponding temperature is shown. Decide tube placement based on this information. Press exit twice to return on the main menu. Select “Files” then “Load,” then “Gradient.” If cells are being used, set the cell lysing step (step 1) to 1 minute (1:00); if purified DNA is being used, set this time to 1 second (0:01). Step 2 should be 94C, Step 4 should be 72˚C, and the lid temperature should be 105˚C. Go to step 3 and set an annealing temperature for the center column. Leave the next two lines as they are, and change the gradient setting (“G”) to the +/- the center temperature amount. Press exit. If prompted to save, press enter three times; if not prompted to save, press enter once. Press exit to get back to the main menu. To begin cycle, select “Start,” then select “Gradient.” The program should begin. <br><br />
<br />
5. The thermocycler is set to store the completed reaction tubes at 4˚C when finished. <br><br />
<br />
=== Ligation ===<br />
<br />
Ligation is the process by which the insert (target DNA gene) is inserted into a plasmid. Both the plasmid and insert have been digested and have the proper “sticky” or blunt ends which are compatible for joining the two DNA pieces together into one molecule. These two DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid. This newly formed plasmid may then be isolated using gel electrophoresis and then used for bacterial transformation or other experimentation. The following is the procedure used by USU to ligate PHB genes into the biobrick plasmids. <br><br />
'''Method'''<br><br />
1. Obtain the following reagents, some of which are in the -20˚C freezer: DNA vector, DNA insert, 10X ligation buffer, T4 DNA ligase (take out only when needed, then return immediately to freezer), and sterile distilled water.<br><br />
2. Ideally, it is desirable to have the concentration of insert ends (or moles of insert) be two to three times the concentration of vector ends (or moles of vector), with a total DNA concentration of 50-400 ng/μl in the reaction. If determining the DNA concentration is not possible, place two to three times the volume of vector as the volume of insert in the reaction. As this is often the case, place the following reagents in a thin-walled PCR tube in the following volumes:<br><br />
<br />
• 10 μl insert DNA<br><br />
• 3 μl vector DNA<br><br />
• 2 μl 10X ligation buffer<br><br />
• 4 μl H2O<br><br />
• 1 μl T4 DNA ligase<br><br />
= ''20 μl total''<br><br />
<br />
This could also be done in different volumes depending on DNA concentration/total volume desired.<br><br />
3. Gently mix the tube, and place the tube in the PCR thermocyler, turn on the machine, select “Start,” from the main menu, select “22” and press “Start.” The thermocycler will keep the reaction at 22˚C.<br><br />
4. Incubate for 60 minutes. Heat-inactivate by placing tubes in 68C water bath for 10 minutes. Place in the freezer if storing for later use. <br></div>Liblinthttp://2008.igem.org/Team:Utah_State/ProtocolsTeam:Utah State/Protocols2008-10-30T03:40:12Z<p>Liblint: /* Restriction Enzyme Digestion and Electrophoresis */</p>
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<br />
=== Bacterial Transformation === <br />
<br />
[[Image:TrentLab1.jpg|300px|right]]<br />
Once the target DNA has been successfully ligated into the plasmid vector, the plasmid must be transferred into the host cell for replication and cloning. In order to do this, the bacterial cells must first be made “competent.” The term “competent” is to describe a cell state in which there exist gaps or openings in the cell wall which will allow the plasmid containing the target genes to enter into the cell. Several methods to make bacterial cells competent exist, such as the calcium chloride method and electroporation. Competent cells may also be purchased commercially. The team at USU has purchased competent cells for all experiments. The following is the method used by the USU team to insert the plasmids containing the PHB biobricks into the cells. <br />
''Trent Mortensen is shown in the right image inspecting agar plates containing transforming cells''<br />
<br />
'''Method'''<br><br />
1. Ensure the necessary antibiotic agar plates have been prepared or begin their preparation now. Four plates per transformation will be necessary (two today, then two tomorrow for streaking). Also ensure that 10 ml liquid media is made up per transformation (also for tomorrow).<br><br />
2. Clean punchout tool by dipping in 10% chlorox, deionized water, deionized water again, then 80%aq ethanol. Let dry for 2 minutes and repeat cleaning procedure between punchouts. <br><br />
3. Punch out gene of choice with a twisting motion, allowing the metal to cut the paper. Use the center part of the punchout tool to dislodge the paper into a 2.5 ml microcentrifuge tube. <br> <br />
4. Add 5 μl TE buffer, place in a 42˚C water bath, and allow plasmids in the paper to elute for 20 minutes.<br />
5. Centrifuge tube(s) at 15K RPM for 3 minutes. Remove SOC media from the -20˚C freezer and leave out to thaw.<br><br />
6. Take competent cells from the -80˚C freezer and place on an ice bath.<br><br />
7. Pipette contents of tube up and down a few times then take 2 μl of the DNA solution and add to the competent cells. Ensure the pipetting is done directly into the cell solution. Let cells incubate in the ice bath for 30 minutes. Heat water bath to 42˚C.<br><br />
8. Heat shock cells in the 42˚C water bath for 30 seconds. Remove and place back in the ice bath for 2 minutes.<br><br />
9. In the hood, add 250 μl SOC media to each tube, bringing the total cell solution to 300 μl. Incubate at 37˚C for 1 hour.<br><br />
10. Get out the antibiotic agar plates. In the hood, add 200 μl of each transformed cell solution to the appropriate antibiotic plate. Use the Bunsen burner to create a “hockey stick” out of a glass pipette tip by holding over the flame until it bends. Allow to cool. Spread cell solution uniformly over the agar plate using the “hockey stick,” then before discarding, spread residual solution on the “stick” over a second plate to get more a more sparse colony distribution. <br><br />
11. Parafilm all plates and place in 37˚C incubator 12-14 hours, or overnight if that is not possible. <br><br />
<br />
=== Streak Plates and Liquid Cultures from Transformed Colonies ===<br />
<br />
After bacterial cells have been transformed, successfully transformed cells must be selected. Because 100% of the cells do not receive the desired plasmid and target gene, it is essential to select for cells that do have the target genes. Several methods are used to accomplish this, such as incorporation of antibiotic resistance and also the lac operon. The USU team has used antibiotic resistance to select for successful transformations. To do this, an antibiotic resistance gene is also added to the plasmid vector that contains the target genes. By doing so, it is possible to know that a cell was successfully transformed based on its ability to grow on an agar plate with antibiotics added. Because the cell is able to grow, the antibiotic resistance gene must be present as well as the target gene. From the agar plates containing the antibiotics, a colony is picked off and transferred into a liquid culture for further analysis and cellular production. The following is the method used by USU to clone the DNA and select for the successful transformation of the PHB biobrick into the cells.<br />
<br />
'''Method'''<br><br />
1. Prepare two 12 ml tubes per transformation, each with 5 ml media containing the appropriate antibiotic (if felt necessary). Get out antibiotic agar plates. Inspect plates from yesterday for colonies. At least two colonies are preferred, but one will do. Select two colonies and label them. <br><br />
2. Use a pipette tip to extract half of each colony and inoculate one agar plate per colony. Using a pipette with a tip, extract the other half of each colony and inoculate one liquid media tube per colony. Label all tubes and plates and place in the 37˚C incubator until tomorrow morning. <br><br />
<br />
=== Preparation for DNA Separation ===<br />
[[Image:Microcentrifuge.JPG|300px|right]]<br />
Following successful bacterial cloning and isolation, it is important to verify that the target gene is in the cell and that the plasmid is functional. To do this, it is a common practice to sequence the DNA plasmid. To obtain enough DNA for sequencing, the bacterial clones are grown in a liquid culture. The cells are harvested by centrifugation and then prepared for DNA plasmid extraction. DNA plasmid extraction can be done several ways, and the overall purpose is to lyse the cells and separate the plasmid DNA from all other cellular proteins, DNA, and debris. The following is the method used by the USU team to isolate the plasmid DNA containing the PHB biobricks. ''An Eppendorf Microcentrifuge was used in these experiments and is shown on the right.''<br />
<br />
'''Method'''<br><br />
1. Prepare two water baths, one boiling and the other 68˚C. <br> <br />
2. Centrifuge the 12 ml tubes containing the 5 ml cultures in the large centrifuge at 8K RPM for 20 min. Discard supernatant. <br> <br />
3. Resuspend cells in 200 μl of STET buffer. <br> <br />
4. Add 10 μl (if older preparation) Lysozyme (50 mg/ml) and incubate at room temperature for 5 min. <br> <br />
5. Boil for 45 sec and centrifuge for 20 min at 13K RPM (or until pellet gets tight).<br> <br />
6. Use a pipette tip to remove the pellet.<br> <br />
7. Add 5 μl RNase A (10 mg/ml) and incubate at 68˚C for 10 minutes.<br> <br />
8. Add 10 μl of 5% CTAB and incubate at room temperature for 3 min.<br> <br />
9. Centrifuge for 5 min at 13K RPM, discard supernatant, and resuspend in 300 μl of 1.2 M NaCl using a vortex mixer.<br> <br />
10. Add 750 μl of ethanol and centrifuge for 5 min at 13K RPM.<br> <br />
11. Discard supernatant, rinse pellet (cannot see) in 80% ethanol, and let tubes dry upside down with caps open.<br><br />
<br />
=== Restriction Enzyme Digestion and Electrophoresis ===<br />
<br />
Restriction enzyme digestion is the process by which an insert DNA sequence is separated from the rest of the DNA molecule. Specific knowledge of the DNA insert is needed to determine which enzyme and conditions to use during the digestion reaction. Once the DNA sequence is known and the correct enzymes have been selected, the DNA may be digested. Listed below is the procedure used by USU to digest the cellular DNA. After enzyme digestion, electrophoresis is used to separate the plasmid from the insert. A gel is prepared and the respective reaction mixes are loaded into the gel. Using a DNA ladder, and knowing the size of the insert, the corresponding band can be seen and cut out of the gel. The insert may then be removed and isolated from the gel, thus yielding the desired DNA. The DNA from this may then be used in PCR reactions, sequencing, ligations for further experimentation, and many more. Listed below are the protocols used by the USU team to run the electrophoresis reaction. <br />
<br />
'''Method'''<br><br />
1. Resuspend DNA in 40 μl water, vortex, and do a brief centrifuge to get solution to the bottom of the tube. <br><br />
2. Add components to the digestion solution in the following order: DNA (23 μl), 10X Tango buffer (3 μl), Xba1 (2 μl), and Pst1 (2 μl). The volume and restriction enzymes can be varied, but it should be ensured that the total volume is 10X the amount of Tango buffer added. Tap tubes periodically and allow to digest while preparing electrophoresis gel. <br><br />
3. Prepare electrophoresis gel by adding 2 g agarose to 200 ml TAE (1% solution). This is best done in an Erlenmeyer flask of adequate volume as swirling will need to be done. Place in the microwave and microwave on high for 20 seconds at a time, pulling it out and swirling until solution is homogeneous again, then repeating (BE CAREFUL to watch the solution closely when swirling – it superheats and can boil over and cause severe burns). Continue until solution is seen boiling in the microwave then gently swirl again. <br><br />
4. Add 20 μl ethidium bromide to solution and swirl until dissolved evenly. <br><br />
5. Add 6 μl of 6X loading dye to each tube of digested DNA solution. <br><br />
6. Prepare the electrophoresis unit by orienting the basin sideways with rubber gaskets firmly against the side. Place desired well template in the basin. <br><br />
7. When the agarose solution is cool enough to comfortably touch the flask, pour into the basin until the solution is about ¾ of the way to the top of the well template. <br><br />
8. When the gel is solidified (should look somewhat cloudy), remove the well template and change basin orientation to have the wells closest to the negative pole (as the DNA will flow towards the positive pole). Pour 1X TAE buffer into both sides of the electrophoresis unit until it just covers the gel and fills the wells.<br><br />
9. By inserting the pipette tip below the TAE liquid and into the well, add 10 μl of DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the digested DNA solutions to the wells by adding about 2 μl less than the total volume in the tubes to prevent air bubbles in the wells.<br><br />
10. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 70 V (this can be as high as 100 V if time is an issue), and press the start button. Separation should take two to three hours. The yellow dye shows the location of the smaller nucleotide lengths and the blue dye shows the location of the larger nucleotide lengths. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. The basin can then be placed back in the electrophoresis unit for further separation if desired. Take care to not have the power supply on without the lid to the unit in place. <br><br />
11. When the desired level of separation is attained, the basin can be placed on the transilluminator for picture taking. Place the cone-shaped cover over the transilluminator and place the digital camera in the top hole for pictures.<br><br />
<br />
''The electrophoresis units and UV transilluminator used in this project are shown in the left and center images below. An electrophoresis image is given on the right.''<br />
<br />
[[Image:Electrophoresis_Units.JPG|320px]][[Image:UV_Transilluminator.JPG|320px]][[Image:Elect.jpg|320px]]<br />
<br />
=== Media Preparation ===<br />
<br />
For all experimentation involving the need for bacterial biomass and experimentation, proper media is needed to grow the cells. The following is the media composition and methods used by USU to prepare the media.<br />
<br />
1. Add 5 g yeast extract, 10 g NaCl, 10 g Bacto Tryptone, and 15 g agar (if desired) to a 2 L Erlenmeyer flask and bring the volume up to 1 L with ddH20. Mix by swirling. Cover top with foil.<br><br />
2. Autoclave for 45 minutes (liquid setting, 0 minutes drying time). It will take an additional half hour for the autoclave to finish cooling then an additional 20 to 30 minutes until the media is cool enough to pour.<br><br />
<br />
=== 1X TAE Preparation ===<br />
1. Add 40 ml 50X TAE solution to a 2 L flask and bring level up to 2 L with ddH20. <br><br />
<br />
=== Polymerase Chain Reaction (PCR) ===<br />
[[Image:Thermocycler.JPG|250px|right]]<br />
<br />
When large amounts of DNA are needed it is necessary to do a PCR reaction. When a PCR reaction is set up and operated properly it will amplify a single target sequence of DNA. The reaction is first set up by designing primers to use that will bind only to the desired regions of the DNA sequence to be replicates and choosing a polymerase to copy the DNA. Once the primer and polymerase has been chosen, the reaction conditions of time and temperature must be optimized. Thus, when the reaction works properly only the target DNA will be amplified into large quantities that may then be isolated and used for further experimentation. The following is the procedure used by USU for PCR reactions to amplify the PHB synthesis and promotion genes.<br> ''An Eppendorf Thermocycle was used in these experiments, as shown in the image on the right.''<br />
<br />
'''Method'''<br><br />
1. Obtain the following reagents from the freezer: DNA template (cells or DNA), 10X Taq buffer (+KCl, -Mg/Cl<small>2</small>), MgCl<small>2</small>, 10 mM dNTP Mix, Taq polymerase (take out of freezer only immediately when needed and put back), and sterile distilled H<small>2</small>O. Place all reagents on ice. Also obtain PCR (either 0.2 or 0.5 ml) tubes.<br><br />
2. Add the following reagents to a tube (50 μl reaction) in the following volumes and order:<br><br />
• 32 μl sterile H<small>2</small>O,<br><br />
• 5 μl 10X buffer, <br><br />
• 2 μl dNTP Mix, <br><br />
• 6 μl MgCl<small>2</small><br><br />
• 3 μl cells/DNA, <br><br />
• 0.25 μl Taq Polymerase<br><br />
• 1 μl primer 1<br><br />
• 1 μl primer 2<br><br />
MgCl<small>2</small> volume can be varied (lower to increase specificity – just ensure total volume is 50 ul with H<small>2</small>O). If many reactions are to be constructed, a master mix can be made up to cut down on time and pipette tip usage (if this is done, ensure primers are added to the appropriate reaction, i.e. perhaps not to the master mix). Tap or vortex tubes and take to the thermocyler. Place all reagents back in the -20˚C freezer. <br><br />
3. Choose thermocycler temperatures. The Eppendorf Mastercycler will cycle between three temperatures: typical temperatures are 94˚C for denaturing, 50-60˚C for primer annealing, and 72˚C for polymerase extending. Lowering the annealing temperature decreases DNA specificity; 55˚C is a good temperature to begin if no trials have been made with the sample. <br><br />
4. Turn on thermocycler with the switch in the back of the unit and open the lid. The placement of the tubes depends on the size of the tube (0.2 or 0.5 ml) and whether or not a temperature gradient is to be used. <br><br><br />
<br />
* If no gradient will be used, tubes can be placed anywhere on the unit in the appropriately-sized hole. Select “Files” and press enter. Select “Load” and then “Standard.” If cells will be used in the reaction, include a 1-minute lysing step at the beginning (step 1); this will be followed by a 1-minute DNA denaturing step (step 2). If purified DNA will be used, set step 1 to 1 second. Set an annealing temperature for step 3. Ensure the lid temperature is 105˚C and the extending temperature is 72˚C. Press exit. If prompted to save, save by pressing enter three times. Press exit to return to the main menu. Choose “Start” on the main menu and select “Standard.” The program should begin. <br><br />
<br />
* If a gradient is to be used, temperature will vary according to column. A 20˚C range is the maximum range that can be used (+/- 10˚C). The range is made by setting a temperature for the middle column and then setting a +/- range. To see what the temperatures will be if a gradient is used, select “OPTIONS” on the main menu, then select “Gradient.” Select the size tube that is being used by pressing “Sel,” then press enter. Choose a temperature for the center column, press enter, then select a +/- range and press enter. The column number along with the corresponding temperature is shown. Decide tube placement based on this information. Press exit twice to return on the main menu. Select “Files” then “Load,” then “Gradient.” If cells are being used, set the cell lysing step (step 1) to 1 minute (1:00); if purified DNA is being used, set this time to 1 second (0:01). Step 2 should be 94C, Step 4 should be 72˚C, and the lid temperature should be 105˚C. Go to step 3 and set an annealing temperature for the center column. Leave the next two lines as they are, and change the gradient setting (“G”) to the +/- the center temperature amount. Press exit. If prompted to save, press enter three times; if not prompted to save, press enter once. Press exit to get back to the main menu. To begin cycle, select “Start,” then select “Gradient.” The program should begin. <br><br />
<br />
5. The thermocycler is set to store the completed reaction tubes at 4˚C when finished. <br><br />
<br />
=== Ligation ===<br />
<br />
Ligation is the process by which the insert (target DNA gene) is inserted into a plasmid. Both the plasmid and insert have been digested and have the proper “sticky” or blunt ends which are compatible for joining the two DNA pieces together into one molecule. These two DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid. This newly formed plasmid may then be isolated using gel electrophoresis and then used for bacterial transformation or other experimentation. The following is the procedure used by USU to ligate PHB genes into the biobrick plasmids. <br><br />
'''Method'''<br><br />
1. Obtain the following reagents, some of which are in the -20˚C freezer: DNA vector, DNA insert, 10X ligation buffer, T4 DNA ligase (take out only when needed, then return immediately to freezer), and sterile distilled water.<br><br />
2. Ideally, it is desirable to have the concentration of insert ends (or moles of insert) be two to three times the concentration of vector ends (or moles of vector), with a total DNA concentration of 50-400 ng/μl in the reaction. If determining the DNA concentration is not possible, place two to three times the volume of vector as the volume of insert in the reaction. As this is often the case, place the following reagents in a thin-walled PCR tube in the following volumes:<br><br />
<br />
• 10 μl insert DNA<br><br />
• 3 μl vector DNA<br><br />
• 2 μl 10X ligation buffer<br><br />
• 4 μl H2O<br><br />
• 1 μl T4 DNA ligase<br><br />
= ''20 μl total''<br><br />
<br />
This could also be done in different volumes depending on DNA concentration/total volume desired.<br><br />
3. Gently mix the tube, and place the tube in the PCR thermocyler, turn on the machine, select “Start,” from the main menu, select “22” and press “Start.” The thermocycler will keep the reaction at 22˚C.<br><br />
4. Incubate for 60 minutes. Heat-inactivate by placing tubes in 68C water bath for 10 minutes. Place in the freezer if storing for later use. <br></div>Liblinthttp://2008.igem.org/Team:Utah_State/TeamTeam:Utah State/Team2008-10-30T03:37:18Z<p>Liblint: </p>
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== '''iGEM 2008 at USU''' ==<br />
[[Image:USU_iGEM.jpg|500px|right]]<br />
|'''TEAM USU BACKGROUND'''<br><br />
Utah State University is proud to be involved in the 2008 iGEM competition for its first year. The 2008 USU iGEM team consists of 4 graduate, 5 undergraduate, and 2 high school students under the supervision of faculty with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Though many project topics were seriously discussed, the team chose to study a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB operon. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction.<br />
<br />
== '''Team Members''' ==<br />
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'''FACULTY ADVISORS:'''<br />
*'''Scott Hinton''': Dean of the College of Engineering, USU<br />
*'''Dr. Charles Miller''': Department of Biological and Irrigation Engineering, USU<br />
*'''Dr. Ronald C. Sims''': Department of Biological and Irrigation Engineering, USU<br />
<br />
'''GRADUATE STUDENTS:''' <br />
*'''Junling Huo''': PhD student, Department of Biological and Irrigation Engineering, USU<br />
*'''Steven Merrigan''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Joseph Camire''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Kirsten Sims''': MS student, Department of Biological and Irrigation Engineering, USU <br />
<br />
'''UNDERGRADUATE STUDENTS:'''<br />
*'''Trent Mortensen''': Department of Biological and Irrigation Engineering, USU <br />
*'''Elisabeth Linton''': Department of Biological and Irrigation Engineering, USU <br />
*'''Daniel Nelson''': Department of Biological and Irrigation Engineering, USU <br />
*'''Rachel Porter''': Department of Biological and Irrigation Engineering, USU <br />
<br />
'''HIGH SCHOOL STUDENTS:'''<br />
*'''Garrett Hinton''': Sky View High School<br />
*'''Matthew Sims''': Logan High School<br />
|<br />
<gallery><br />
Image:Scott_Hinton.jpg|Dean Scott Hinton<br />
Image:Charles-Miller.jpg|Dr. Charlie Miller<br />
Image:Ron_sims.jpg|Dr. Ron Sims<br />
Image:JH.jpg|Junlng Huo<br />
Image:JC.jpg|Joseph Camire<br />
Image:Stephen_Merrigan.JPG|Stephen Merrigan<br />
Image:Libby_linton.jpg|Elisabeth Linton<br />
Image:DN.jpg|Daniel Nelson<br />
Image:rachel_249(2).jpg|Rachel Porter<br />
Image:TM.jpg|Trent Mortensen<br />
Image:Sims.jpg|Kirsten and Matthew Sims<br />
Image:GH.jpg|Garrett Hinton<br />
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== '''Team Member Contributions''' ==<br />
*'''Junling Huo''' is a PhD student in Biological Engineering who served as an advisor for the iGEM team. He helped to answer student questions, and guide such experimental activities as primer design, electrophoresis, and DNA purification. His personal research is on ''Rhodobacter sphaeroides'', a photosynthetic bacterium.<br />
<br />
*'''Stephen Merrigan''' is currently working on a MS in Biological Engineering at Utah State studying biodiesel production from algae, and has a background in Microbiology. Stephen's primary role was as an advisor for the project. During the first few months of the project Stephen did background research on project topics. He also helped with lab work when necessary, including DNA purification. Stephen also advised on the wiki construction.<br />
<br />
*'''Joseph Camire''' advised on targeting the amplification of the full phaCAB cassette and phaC gene,optimizing the PCR process and testing of primer sets with and without prefix and suffix regions. His work also involved sequence determination and analysis of the phaCAB cassette contained in the source plasmid. He also guided work on the phaC gene, which included targeting at amplification and preparing the gene for site-directed mutagenesis for removal of a critical restriction enzyme site prior to ligation of a new phaCAB cassette for biobrick construction.<br />
<br />
*'''Kirsten Sims''' is a first-year graduate student at Utah State University in Biological and Irrigation Engineering. She received her Bacherlor's degree from Gonzaga University in Biology. Her research focuses on the development of cellulose-derived biofuel. As a member of the USU iGEM team, she was involved in the development and design of the project, as well as participation in the laboratory procedures. She also contributed to the development of the wiki by providing an abstract of the project.<br />
<br />
*'''Trent Mortensen''' is a finishing senior in Biological Engineering. His main research direction is biomedical in nature, using herbal antibiotics as possible alternatives for disease treatment. As part of the USU iGEM team, he played an active role in the planning of the project, group coordination, advising professor consultation, laboratory work, ordering of materials, lab maintenance, Wiki preparation, documentation, and presentation preparations throughout the course of the project.<br />
<br />
*'''Elisabeth Linton''' is a Biological Engineering student. Her research is focused on the the analysis and verification of gas chromatography and nuclear magnetic resonance methods for the quantification of intracellular polyhydroxyalkanoates in bacterial and environmental samples. For the iGEM team, Libbie helped in project determination and planning, as well as literature review and topic research. She also carried out work in the laboratory like restriction enzyme digestions, gel electrophoresis, DNA isolation, and bacterial transformations. She was also responsible for coordinating efforts and organizing the wiki.<br />
<br />
*'''Daniel Nelson''' is pursuing a degree in Biological Engineering and his current personal research project is “Omega-3 Fatty Acid Production and Extraction in Schizochytrium limacinum SR21.” He has enjoyed participating in group discussions as project ideas and direction have been determined. In the lab, he has worked with the team to isolate, purify, and analyze the key DNA promoters for the PHB synthesis gene. He hopes that through study of these promoter regions, the team will find a way to increase PHB production and create a general gene expression system to monitor cellular product accumulation. Outside of the lab, he has helped to design the logo for the USU team.<br />
<br />
*'''Rachel Porter''' is a sophomore at Utah State University majoring in Biological Engineering/pre-med. For the iGEM project, she helped to carry out some of the lab work. In the lab, she helped isolate and purify DNA, prepare and run electrophoresis gels, and culture cells.<br />
<br />
*'''Matthew Sims''' is a Senior attending Logan High School. This is his first year participating in laboratory research at Utah State University. He plans to go to college next fall and continue his studies in molecular biology and biochemistry. He helped during the summer months to carry out many experiments and obtain and analyze data.<br />
<br />
*'''Garrett Hinton''' is a junior at Sky View High School in Smithfield, Utah. He was heavily involved in the project discussion meetings, as well as the background literature research. Garrett spent many hours working on this project and carried out a substantial portion of the laboratory work.<br />
<br />
'''''We would like to thank our faculty advisers - Dr. Ron Sims, Dean Scott Hinton, and Dr. Charlie Miller - for all of their support and for providing this opportunity for the students.'''''<br />
[[Image:Advisors.jpg|350px|center]]<br />
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== '''Logan and USU''' ==<br />
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<b><a href="http://www.usu.edu/">Utah State University</a></b> is located in Logan, Utah. Logan is about 85 miles north of Salt Lake City, Utah. The city of Logan is located in the heart of Cache Valley near the on the western slopes of the Bear River Mountains, the northernmost branch of the Wasatch Range. The city has a population of approximately 47,000. Logan was established in 1859 and has a rich heritage and wonderful culture. The city of Logan has been stated to be among the safest cities in America.<br><br />
<br><br />
Utah State University was established in 1888 as the Agricultural College of Utah. It's name was later changed to Utah State Agricultural College and was again changed to Utah State University (USU) in 1957. As the land-grant university in Utah, USU conducts world-class research in a variety of agricultural and natural resource disciplines, and has several projects in conjunction with the Department of Defense, NASA. Utah State University also conducts extensive aerospace research. The main campus is located in Logan, Utah. Beyond the Logan campus, Utah State's Extension programs extend academic resources and support throughout the entire state of Utah, having extension locations in each of Utah's 29 counties.<br><br />
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''The Sant Building, shown below, is home to the new USU synthetic biology laboratory''<br />
[[Image:Sant_Blgd.jpg|600px|center]]</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProtocolsTeam:Utah State/Protocols2008-10-30T03:35:54Z<p>Liblint: /* Preparation for DNA Separation */</p>
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<!--- The Mission, Experiments ---><br />
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=== Bacterial Transformation === <br />
<br />
[[Image:TrentLab1.jpg|300px|right]]<br />
Once the target DNA has been successfully ligated into the plasmid vector, the plasmid must be transferred into the host cell for replication and cloning. In order to do this, the bacterial cells must first be made “competent.” The term “competent” is to describe a cell state in which there exist gaps or openings in the cell wall which will allow the plasmid containing the target genes to enter into the cell. Several methods to make bacterial cells competent exist, such as the calcium chloride method and electroporation. Competent cells may also be purchased commercially. The team at USU has purchased competent cells for all experiments. The following is the method used by the USU team to insert the plasmids containing the PHB biobricks into the cells. <br />
''Trent Mortensen is shown in the right image inspecting agar plates containing transforming cells''<br />
<br />
'''Method'''<br><br />
1. Ensure the necessary antibiotic agar plates have been prepared or begin their preparation now. Four plates per transformation will be necessary (two today, then two tomorrow for streaking). Also ensure that 10 ml liquid media is made up per transformation (also for tomorrow).<br><br />
2. Clean punchout tool by dipping in 10% chlorox, deionized water, deionized water again, then 80%aq ethanol. Let dry for 2 minutes and repeat cleaning procedure between punchouts. <br><br />
3. Punch out gene of choice with a twisting motion, allowing the metal to cut the paper. Use the center part of the punchout tool to dislodge the paper into a 2.5 ml microcentrifuge tube. <br> <br />
4. Add 5 μl TE buffer, place in a 42˚C water bath, and allow plasmids in the paper to elute for 20 minutes.<br />
5. Centrifuge tube(s) at 15K RPM for 3 minutes. Remove SOC media from the -20˚C freezer and leave out to thaw.<br><br />
6. Take competent cells from the -80˚C freezer and place on an ice bath.<br><br />
7. Pipette contents of tube up and down a few times then take 2 μl of the DNA solution and add to the competent cells. Ensure the pipetting is done directly into the cell solution. Let cells incubate in the ice bath for 30 minutes. Heat water bath to 42˚C.<br><br />
8. Heat shock cells in the 42˚C water bath for 30 seconds. Remove and place back in the ice bath for 2 minutes.<br><br />
9. In the hood, add 250 μl SOC media to each tube, bringing the total cell solution to 300 μl. Incubate at 37˚C for 1 hour.<br><br />
10. Get out the antibiotic agar plates. In the hood, add 200 μl of each transformed cell solution to the appropriate antibiotic plate. Use the Bunsen burner to create a “hockey stick” out of a glass pipette tip by holding over the flame until it bends. Allow to cool. Spread cell solution uniformly over the agar plate using the “hockey stick,” then before discarding, spread residual solution on the “stick” over a second plate to get more a more sparse colony distribution. <br><br />
11. Parafilm all plates and place in 37˚C incubator 12-14 hours, or overnight if that is not possible. <br><br />
<br />
=== Streak Plates and Liquid Cultures from Transformed Colonies ===<br />
<br />
After bacterial cells have been transformed, successfully transformed cells must be selected. Because 100% of the cells do not receive the desired plasmid and target gene, it is essential to select for cells that do have the target genes. Several methods are used to accomplish this, such as incorporation of antibiotic resistance and also the lac operon. The USU team has used antibiotic resistance to select for successful transformations. To do this, an antibiotic resistance gene is also added to the plasmid vector that contains the target genes. By doing so, it is possible to know that a cell was successfully transformed based on its ability to grow on an agar plate with antibiotics added. Because the cell is able to grow, the antibiotic resistance gene must be present as well as the target gene. From the agar plates containing the antibiotics, a colony is picked off and transferred into a liquid culture for further analysis and cellular production. The following is the method used by USU to clone the DNA and select for the successful transformation of the PHB biobrick into the cells.<br />
<br />
'''Method'''<br><br />
1. Prepare two 12 ml tubes per transformation, each with 5 ml media containing the appropriate antibiotic (if felt necessary). Get out antibiotic agar plates. Inspect plates from yesterday for colonies. At least two colonies are preferred, but one will do. Select two colonies and label them. <br><br />
2. Use a pipette tip to extract half of each colony and inoculate one agar plate per colony. Using a pipette with a tip, extract the other half of each colony and inoculate one liquid media tube per colony. Label all tubes and plates and place in the 37˚C incubator until tomorrow morning. <br><br />
<br />
=== Preparation for DNA Separation ===<br />
[[Image:Microcentrifuge.JPG|300px|right]]<br />
Following successful bacterial cloning and isolation, it is important to verify that the target gene is in the cell and that the plasmid is functional. To do this, it is a common practice to sequence the DNA plasmid. To obtain enough DNA for sequencing, the bacterial clones are grown in a liquid culture. The cells are harvested by centrifugation and then prepared for DNA plasmid extraction. DNA plasmid extraction can be done several ways, and the overall purpose is to lyse the cells and separate the plasmid DNA from all other cellular proteins, DNA, and debris. The following is the method used by the USU team to isolate the plasmid DNA containing the PHB biobricks. ''An Eppendorf Microcentrifuge was used in these experiments and is shown on the right.''<br />
<br />
'''Method'''<br><br />
1. Prepare two water baths, one boiling and the other 68˚C. <br> <br />
2. Centrifuge the 12 ml tubes containing the 5 ml cultures in the large centrifuge at 8K RPM for 20 min. Discard supernatant. <br> <br />
3. Resuspend cells in 200 μl of STET buffer. <br> <br />
4. Add 10 μl (if older preparation) Lysozyme (50 mg/ml) and incubate at room temperature for 5 min. <br> <br />
5. Boil for 45 sec and centrifuge for 20 min at 13K RPM (or until pellet gets tight).<br> <br />
6. Use a pipette tip to remove the pellet.<br> <br />
7. Add 5 μl RNase A (10 mg/ml) and incubate at 68˚C for 10 minutes.<br> <br />
8. Add 10 μl of 5% CTAB and incubate at room temperature for 3 min.<br> <br />
9. Centrifuge for 5 min at 13K RPM, discard supernatant, and resuspend in 300 μl of 1.2 M NaCl using a vortex mixer.<br> <br />
10. Add 750 μl of ethanol and centrifuge for 5 min at 13K RPM.<br> <br />
11. Discard supernatant, rinse pellet (cannot see) in 80% ethanol, and let tubes dry upside down with caps open.<br><br />
<br />
=== Restriction Enzyme Digestion and Electrophoresis ===<br />
<br />
Restriction enzyme digestion is the process by which a target DNA sequence is separated from the rest of the DNA molecule. Specific knowledge of the target DNA gene is needed to determine which enzyme and conditions to use during the digestion reaction. Once the target DNA sequence is known and the correct enzymes have been selected, the DNA may be digested. Listed below is the procedure used by USU to digest the cellular DNA and obtain the target DNA of PHB synthesis and gene promotion. After enzyme digestion, electrophoresis is used to separate the plasmid DNA from the target DNA genes. A gel is prepared and the respective reaction mixes are loaded into the gel. Using a DNA ladder, and knowing the size of the target DNA gene, the corresponding band can be seen and cut out of the gel. The target DNA may then be removed and isolated from the gel, thus yielding the desired target DNA genes. The DNA from this may then be used in PCR reactions, sequencing, ligations for further experimentation, and many more. Listed below are the protocols used by the USU team to run the electrophoresis reaction. <br />
<br />
'''Method'''<br><br />
1. Resuspend DNA in 40 μl water, vortex, and do a brief centrifuge to get solution to the bottom of the tube. <br><br />
2. Add components to the digestion solution in the following order: DNA (23 μl), 10X Tango buffer (3 μl), Xba1 (2 μl), and Pst1 (2 μl). The volume and restriction enzymes can be varied, but it should be ensured that the total volume is 10X the amount of Tango buffer added. Tap tubes periodically and allow to digest while preparing electrophoresis gel. <br><br />
3. Prepare electrophoresis gel by adding 2 g agarose to 200 ml TAE (1% solution). This is best done in an Erlenmeyer flask of adequate volume as swirling will need to be done. Place in the microwave and microwave on high for 20 seconds at a time, pulling it out and swirling until solution is homogeneous again, then repeating (BE CAREFUL to watch the solution closely when swirling – it superheats and can boil over and cause severe burns). Continue until solution is seen boiling in the microwave then gently swirl again. <br><br />
4. Add 20 μl ethidium bromide to solution and swirl until dissolved evenly. <br><br />
5. Add 6 μl of 6X loading dye to each tube of digested DNA solution. <br><br />
6. Prepare the electrophoresis unit by orienting the basin sideways with rubber gaskets firmly against the side. Place desired well template in the basin. <br><br />
7. When the agarose solution is cool enough to comfortably touch the flask, pour into the basin until the solution is about ¾ of the way to the top of the well template. <br><br />
8. When the gel is solidified (should look somewhat cloudy), remove the well template and change basin orientation to have the wells closest to the negative pole (as the DNA will flow towards the positive pole). Pour 1X TAE buffer into both sides of the electrophoresis unit until it just covers the gel and fills the wells.<br><br />
9. By inserting the pipette tip below the TAE liquid and into the well, add 10 μl of DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the digested DNA solutions to the wells by adding about 2 μl less than the total volume in the tubes to prevent air bubbles in the wells.<br><br />
10. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 70 V (this can be as high as 100 V if time is an issue), and press the start button. Separation should take two to three hours. The yellow dye shows the location of the smaller nucleotide lengths and the blue dye shows the location of the larger nucleotide lengths. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. The basin can then be placed back in the electrophoresis unit for further separation if desired. Take care to not have the power supply on without the lid to the unit in place. <br><br />
11. When the desired level of separation is attained, the basin can be placed on the transilluminator for picture taking. Place the cone-shaped cover over the transilluminator and place the digital camera in the top hole for pictures.<br><br />
<br />
''The electrophoresis units and UV transilluminator used in this project are shown in the left and center images below. An electrophoresis image is given on the right.''<br />
<br />
[[Image:Electrophoresis_Units.JPG|320px]][[Image:UV_Transilluminator.JPG|320px]][[Image:Elect.jpg|320px]]<br />
<br />
=== Media Preparation ===<br />
<br />
For all experimentation involving the need for bacterial biomass and experimentation, proper media is needed to grow the cells. The following is the media composition and methods used by USU to prepare the media.<br />
<br />
1. Add 5 g yeast extract, 10 g NaCl, 10 g Bacto Tryptone, and 15 g agar (if desired) to a 2 L Erlenmeyer flask and bring the volume up to 1 L with ddH20. Mix by swirling. Cover top with foil.<br><br />
2. Autoclave for 45 minutes (liquid setting, 0 minutes drying time). It will take an additional half hour for the autoclave to finish cooling then an additional 20 to 30 minutes until the media is cool enough to pour.<br><br />
<br />
=== 1X TAE Preparation ===<br />
1. Add 40 ml 50X TAE solution to a 2 L flask and bring level up to 2 L with ddH20. <br><br />
<br />
=== Polymerase Chain Reaction (PCR) ===<br />
[[Image:Thermocycler.JPG|250px|right]]<br />
<br />
When large amounts of DNA are needed it is necessary to do a PCR reaction. When a PCR reaction is set up and operated properly it will amplify a single target sequence of DNA. The reaction is first set up by designing primers to use that will bind only to the desired regions of the DNA sequence to be replicates and choosing a polymerase to copy the DNA. Once the primer and polymerase has been chosen, the reaction conditions of time and temperature must be optimized. Thus, when the reaction works properly only the target DNA will be amplified into large quantities that may then be isolated and used for further experimentation. The following is the procedure used by USU for PCR reactions to amplify the PHB synthesis and promotion genes.<br> ''An Eppendorf Thermocycle was used in these experiments, as shown in the image on the right.''<br />
<br />
'''Method'''<br><br />
1. Obtain the following reagents from the freezer: DNA template (cells or DNA), 10X Taq buffer (+KCl, -Mg/Cl<small>2</small>), MgCl<small>2</small>, 10 mM dNTP Mix, Taq polymerase (take out of freezer only immediately when needed and put back), and sterile distilled H<small>2</small>O. Place all reagents on ice. Also obtain PCR (either 0.2 or 0.5 ml) tubes.<br><br />
2. Add the following reagents to a tube (50 μl reaction) in the following volumes and order:<br><br />
• 32 μl sterile H<small>2</small>O,<br><br />
• 5 μl 10X buffer, <br><br />
• 2 μl dNTP Mix, <br><br />
• 6 μl MgCl<small>2</small><br><br />
• 3 μl cells/DNA, <br><br />
• 0.25 μl Taq Polymerase<br><br />
• 1 μl primer 1<br><br />
• 1 μl primer 2<br><br />
MgCl<small>2</small> volume can be varied (lower to increase specificity – just ensure total volume is 50 ul with H<small>2</small>O). If many reactions are to be constructed, a master mix can be made up to cut down on time and pipette tip usage (if this is done, ensure primers are added to the appropriate reaction, i.e. perhaps not to the master mix). Tap or vortex tubes and take to the thermocyler. Place all reagents back in the -20˚C freezer. <br><br />
3. Choose thermocycler temperatures. The Eppendorf Mastercycler will cycle between three temperatures: typical temperatures are 94˚C for denaturing, 50-60˚C for primer annealing, and 72˚C for polymerase extending. Lowering the annealing temperature decreases DNA specificity; 55˚C is a good temperature to begin if no trials have been made with the sample. <br><br />
4. Turn on thermocycler with the switch in the back of the unit and open the lid. The placement of the tubes depends on the size of the tube (0.2 or 0.5 ml) and whether or not a temperature gradient is to be used. <br><br><br />
<br />
* If no gradient will be used, tubes can be placed anywhere on the unit in the appropriately-sized hole. Select “Files” and press enter. Select “Load” and then “Standard.” If cells will be used in the reaction, include a 1-minute lysing step at the beginning (step 1); this will be followed by a 1-minute DNA denaturing step (step 2). If purified DNA will be used, set step 1 to 1 second. Set an annealing temperature for step 3. Ensure the lid temperature is 105˚C and the extending temperature is 72˚C. Press exit. If prompted to save, save by pressing enter three times. Press exit to return to the main menu. Choose “Start” on the main menu and select “Standard.” The program should begin. <br><br />
<br />
* If a gradient is to be used, temperature will vary according to column. A 20˚C range is the maximum range that can be used (+/- 10˚C). The range is made by setting a temperature for the middle column and then setting a +/- range. To see what the temperatures will be if a gradient is used, select “OPTIONS” on the main menu, then select “Gradient.” Select the size tube that is being used by pressing “Sel,” then press enter. Choose a temperature for the center column, press enter, then select a +/- range and press enter. The column number along with the corresponding temperature is shown. Decide tube placement based on this information. Press exit twice to return on the main menu. Select “Files” then “Load,” then “Gradient.” If cells are being used, set the cell lysing step (step 1) to 1 minute (1:00); if purified DNA is being used, set this time to 1 second (0:01). Step 2 should be 94C, Step 4 should be 72˚C, and the lid temperature should be 105˚C. Go to step 3 and set an annealing temperature for the center column. Leave the next two lines as they are, and change the gradient setting (“G”) to the +/- the center temperature amount. Press exit. If prompted to save, press enter three times; if not prompted to save, press enter once. Press exit to get back to the main menu. To begin cycle, select “Start,” then select “Gradient.” The program should begin. <br><br />
<br />
5. The thermocycler is set to store the completed reaction tubes at 4˚C when finished. <br><br />
<br />
=== Ligation ===<br />
<br />
Ligation is the process by which the insert (target DNA gene) is inserted into a plasmid. Both the plasmid and insert have been digested and have the proper “sticky” or blunt ends which are compatible for joining the two DNA pieces together into one molecule. These two DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid. This newly formed plasmid may then be isolated using gel electrophoresis and then used for bacterial transformation or other experimentation. The following is the procedure used by USU to ligate PHB genes into the biobrick plasmids. <br><br />
'''Method'''<br><br />
1. Obtain the following reagents, some of which are in the -20˚C freezer: DNA vector, DNA insert, 10X ligation buffer, T4 DNA ligase (take out only when needed, then return immediately to freezer), and sterile distilled water.<br><br />
2. Ideally, it is desirable to have the concentration of insert ends (or moles of insert) be two to three times the concentration of vector ends (or moles of vector), with a total DNA concentration of 50-400 ng/μl in the reaction. If determining the DNA concentration is not possible, place two to three times the volume of vector as the volume of insert in the reaction. As this is often the case, place the following reagents in a thin-walled PCR tube in the following volumes:<br><br />
<br />
• 10 μl insert DNA<br><br />
• 3 μl vector DNA<br><br />
• 2 μl 10X ligation buffer<br><br />
• 4 μl H2O<br><br />
• 1 μl T4 DNA ligase<br><br />
= ''20 μl total''<br><br />
<br />
This could also be done in different volumes depending on DNA concentration/total volume desired.<br><br />
3. Gently mix the tube, and place the tube in the PCR thermocyler, turn on the machine, select “Start,” from the main menu, select “22” and press “Start.” The thermocycler will keep the reaction at 22˚C.<br><br />
4. Incubate for 60 minutes. Heat-inactivate by placing tubes in 68C water bath for 10 minutes. Place in the freezer if storing for later use. <br></div>Liblinthttp://2008.igem.org/Team:Utah_State/TeamTeam:Utah State/Team2008-10-30T03:34:27Z<p>Liblint: </p>
<hr />
<div>{| align="center"<br />
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|}<br><br />
{|style="font color="#CC3300"; background-color:#212223; cellpadding="3" cellspacing="5" border="2" bordercolor="#cd0000"border-spacing:6px; text-align:center" width="960px"<br />
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#99CCFF">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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<style type="text/css"><br />
body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
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{|align="justify"<br />
== '''iGEM 2008 at USU''' ==<br />
[[Image:USU_iGEM.jpg|500px|right]]<br />
|'''TEAM USU BACKGROUND'''<br><br />
Utah State University is proud to be involved in the 2008 iGEM competition for its first year. The 2008 USU iGEM team consists of 4 graduate, 5 undergraduate, and 2 high school students under the supervision of faculty with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Though many project topics were seriously discussed, the team chose to study a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB operon. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction.<br />
<br />
== '''Team Members''' ==<br />
{|border = "10"<br />
|-<br />
|rowspan="3"|<br />
'''FACULTY ADVISORS:'''<br />
*'''Scott Hinton''': Dean of the College of Engineering, USU<br />
*'''Dr. Charles Miller''': Department of Biological and Irrigation Engineering, USU<br />
*'''Dr. Ronald C. Sims''': Department of Biological and Irrigation Engineering, USU<br />
<br />
'''GRADUATE STUDENTS:''' <br />
*'''Junling Huo''': PhD student, Department of Biological and Irrigation Engineering, USU<br />
*'''Steven Merrigan''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Joseph Camire''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Kirsten Sims''': MS student, Department of Biological and Irrigation Engineering, USU <br />
<br />
'''UNDERGRADUATE STUDENTS:'''<br />
*'''Trent Mortensen''': Department of Biological and Irrigation Engineering, USU <br />
*'''Elisabeth Linton''': Department of Biological and Irrigation Engineering, USU <br />
*'''Daniel Nelson''': Department of Biological and Irrigation Engineering, USU <br />
*'''Rachel Porter''': Department of Biological and Irrigation Engineering, USU <br />
<br />
'''HIGH SCHOOL STUDENTS:'''<br />
*'''Garrett Hinton''': Sky View High School<br />
*'''Matthew Sims''': Logan High School<br />
|<br />
<gallery><br />
Image:Scott_Hinton.jpg|Dean Scott Hinton<br />
Image:Charles-Miller.jpg|Dr. Charlie Miller<br />
Image:Ron_sims.jpg|Dr. Ron Sims<br />
Image:JH.jpg|Junlng Huo<br />
Image:JC.jpg|Joseph Camire<br />
Image:Stephen_Merrigan.JPG|Stephen Merrigan<br />
Image:Libby_linton.jpg|Elisabeth Linton<br />
Image:DN.jpg|Daniel Nelson<br />
Image:rachel_249(2).jpg|Rachel Porter<br />
Image:TM.jpg|Trent Mortensen<br />
Image:Sims.jpg|Kirsten and Matthew Sims<br />
Image:GH.jpg|Garrett Hinton<br />
</gallery><br />
|}<br />
<br />
== '''Team Member Contributions''' ==<br />
*'''Junling Huo''' is a PhD student in Biological Engineering who served as an advisor for the iGEM team. He helped to answer student questions, and guide such experimental activities as primer design, electrophoresis, and DNA purification. His personal research is on ''Rhodobacter sphaeroides'', a photosynthetic bacterium.<br />
<br />
*'''Stephen Merrigan''' is currently working on a MS in Biological Engineering at Utah State studying biodiesel production from algae, and has a background in Microbiology. Stephen's primary role was as an advisor for the project. During the first few months of the project Stephen did background research on project topics. He also helped with lab work when necessary, including DNA purification. Stephen also advised on the wiki construction.<br />
<br />
*'''Joseph Camire''' advised on targeting the amplification of the full phaCAB cassette and phaC gene,optimizing the PCR process and testing of primer sets with and without prefix and suffix regions. His work also involved sequence determination and analysis of the phaCAB cassette contained in the source plasmid. He also guided work on the phaC gene, which included targeting at amplification and preparing the gene for site-directed mutagenesis for removal of a critical restriction enzyme site prior to ligation of a new phaCAB cassette for biobrick construction.<br />
<br />
*'''Kirsten Sims''' is a first-year graduate student at Utah State University in Biological and Irrigation Engineering. She received her Bacherlor's degree from Gonzaga University in Biology. Her research focuses on the development of cellulose-derived biofuel. As a member of the USU iGEM team, she was involved in the development and design of the project, as well as participation in the laboratory procedures. She also contributed to the development of the wiki by providing an abstract of the project.<br />
<br />
*'''Trent Mortensen''' is a finishing senior in Biological Engineering. His main research direction is biomedical in nature, using herbal antibiotics as possible alternatives for disease treatment. As part of the USU iGEM team, he played an active role in the planning of the project, group coordination, advising professor consultation, laboratory work, ordering of materials, lab maintenance, Wiki preparation, documentation, and presentation preparations throughout the course of the project.<br />
<br />
*'''Elisabeth Linton''' is a Biological Engineering student. Her research is focused on the the analysis and verification of gas chromatography and nuclear magnetic resonance methods for the quantification of intracellular polyhydroxyalkanoates in bacterial and environmental samples. For the iGEM team, Libbie helped in project determination and planning, as well as literature review and topic research. She also carried out work in the laboratory like restriction enzyme digestions, gel electrophoresis, DNA isolation, and bacterial transformations. She was also responsible for coordinating efforts and organizing the wiki.<br />
<br />
*'''Daniel Nelson''' is pursuing a degree in Biological Engineering and his current personal research project is “Omega-3 Fatty Acid Production and Extraction in Schizochytrium limacinum SR21.” He has enjoyed participating in group discussions as project ideas and direction have been determined. In the lab, he has worked with the team to isolate, purify, and analyze the key DNA promoters for the PHB synthesis gene. He hopes that through study of these promoter regions, the team will find a way to increase PHB production and create a general gene expression system to monitor cellular product accumulation. Outside of the lab, he has helped to design the logo for the USU team.<br />
<br />
*'''Rachel Porter''' is a sophomore at Utah State University majoring in Biological Engineering/pre-med. For the iGEM project, she helped to carry out some of the lab work. In the lab, she helped isolate and purify DNA, prepare and run electrophoresis gels, and culture cells.<br />
<br />
*'''Matthew Sims''' is a Senior attending Logan High School. This is his first year participating in laboratory research at Utah State University. He plans to go to college next fall and continue his studies in molecular biology and biochemistry. He helped during the summer months to carry out many experiments and obtain and analyze data.<br />
<br />
*'''Garrett Hinton''' is a junior at Sky View High School in Smithfield, Utah. He was heavily involved in the project discussion meetings, as well as the background literature research. Garrett spent many hours working on this project and carried out a substantial portion of the laboratory work.<br />
<br />
'''''We would like to thank our faculty advisers - Dr. Ron Sims, Dean Scott Hinton, and Dr. Charlie Miller - for all of their support and for providing this opportunity for the students.'''''<br />
[[Image:Advisors.jpg|350px|center]]<br />
<br />
== '''Logan and USU''' ==<br />
<html><br />
<style type="text/css"><br />
<br />
</style> <br />
<b><a href="http://www.usu.edu/">Utah State University</a></b> is located in Logan, Utah. Logan is about 85 miles north of Salt Lake City, Utah. The city of Logan is located in the heart of Cache Valley near the on the western slopes of the Bear River Mountains, the northernmost branch of the Wasatch Range. The city has a population of approximately 47,000. Logan was established in 1859 and has a rich heritage and wonderful culture. The city of Logan has been stated to be among the safest cities in America.<br><br />
<br><br />
Utah State University was established in 1888 as the Agricultural College of Utah. It's name was later changed to Utah State Agricultural College and was again changed to Utah State University (USU) in 1957. As the land-grant university in Utah, USU conducts world-class research in a variety of agricultural and natural resource disciplines, and has several projects in conjunction with the Department of Defense, NASA. Utah State University also conducts extensive aerospace research. The main campus is located in Logan, Utah. Beyond the Logan campus, Utah State's Extension programs extend academic resources and support throughout the entire state of Utah, having extension locations in each of Utah's 29 counties.<br><br />
<br />
<br />
</html><br />
[[Image:Sant_Blgd.jpg|600px|center]]</div>Liblinthttp://2008.igem.org/Team:Utah_State/TeamTeam:Utah State/Team2008-10-30T03:32:28Z<p>Liblint: /* iGEM 2008 at USU */</p>
<hr />
<div>{| align="center"<br />
|align="center" |[[Image:USUheader2.gif|960 px]]<br />
|}<br><br />
{|style="font color="#CC3300"; background-color:#212223; cellpadding="3" cellspacing="5" border="2" bordercolor="#cd0000"border-spacing:6px; text-align:center" width="960px"<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State|<font color="#ffffff">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#99CCFF">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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<style type="text/css"><br />
body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
<br />
<br />
</style> <br />
</html><br />
<br />
{|align="justify"<br />
== '''iGEM 2008 at USU''' ==<br />
[[Image:USU_iGEM.jpg|500px|right]]<br />
|'''TEAM USU BACKGROUND'''<br><br />
Utah State University is proud to be involved in the 2008 iGEM competition for its first year. The 2008 USU iGEM team consists of graduate, undergraduate, and high school students with varying levels of experience in genetic and biological engineering under the supervision of faculty with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Though many project topics were seriously discussed, the team chose to study a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction.<br />
<br />
== '''Team Members''' ==<br />
{|border = "10"<br />
|-<br />
|rowspan="3"|<br />
'''FACULTY ADVISORS:'''<br />
*'''Scott Hinton''': Dean of the College of Engineering, USU<br />
*'''Dr. Charles Miller''': Department of Biological and Irrigation Engineering, USU<br />
*'''Dr. Ronald C. Sims''': Department of Biological and Irrigation Engineering, USU<br />
<br />
'''GRADUATE STUDENTS:''' <br />
*'''Junling Huo''': PhD student, Department of Biological and Irrigation Engineering, USU<br />
*'''Steven Merrigan''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Joseph Camire''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Kirsten Sims''': MS student, Department of Biological and Irrigation Engineering, USU <br />
<br />
'''UNDERGRADUATE STUDENTS:'''<br />
*'''Trent Mortensen''': "iGem spelled b-i-e, iGem" <br />
*'''Elisabeth Linton''': Department of Biological and Irrigation Engineering, USU <br />
*'''Daniel Nelson''': Department of Biological and Irrigation Engineering, USU <br />
*'''Rachel Porter''': Department of Biological and Irrigation Engineering, USU <br />
<br />
'''HIGH SCHOOL STUDENTS:'''<br />
*'''Garrett Hinton''': Sky View High School<br />
*'''Matthew Sims''': Logan High School<br />
|<br />
<gallery><br />
Image:Scott_Hinton.jpg|Dean Scott Hinton<br />
Image:Charles-Miller.jpg|Dr. Charlie Miller<br />
Image:Ron_sims.jpg|Dr. Ron Sims<br />
Image:JH.jpg|Junlng Huo<br />
Image:JC.jpg|Joseph Camire<br />
Image:Stephen_Merrigan.JPG|Stephen Merrigan<br />
Image:Libby_linton.jpg|Elisabeth Linton<br />
Image:DN.jpg|Daniel Nelson<br />
Image:rachel_249(2).jpg|Rachel Porter<br />
Image:TM.jpg|Trent Mortensen<br />
Image:Sims.jpg|Kirsten and Matthew Sims<br />
Image:GH.jpg|Garrett Hinton<br />
</gallery><br />
|}<br />
<br />
== '''Team Member Contributions''' ==<br />
*'''Junling Huo''' is a PhD student in Biological Engineering who served as an advisor for the iGEM team. He helped to answer student questions, and guide such experimental activities as primer design, electrophoresis, and DNA purification. His personal research is on ''Rhodobacter sphaeroides'', a photosynthetic bacterium.<br />
<br />
*'''Stephen Merrigan''' is currently working on a MS in Biological Engineering at Utah State studying biodiesel production from algae, and has a background in Microbiology. Stephen's primary role was as an advisor for the project. During the first few months of the project Stephen did background research on project topics. He also helped with lab work when necessary, including DNA purification. Stephen also advised on the wiki construction.<br />
<br />
*'''Joseph Camire''' advised on targeting the amplification of the full phaCAB cassette and phaC gene,optimizing the PCR process and testing of primer sets with and without prefix and suffix regions. His work also involved sequence determination and analysis of the phaCAB cassette contained in the source plasmid. He also guided work on the phaC gene, which included targeting at amplification and preparing the gene for site-directed mutagenesis for removal of a critical restriction enzyme site prior to ligation of a new phaCAB cassette for biobrick construction.<br />
<br />
*'''Kirsten Sims''' is a first-year graduate student at Utah State University in Biological and Irrigation Engineering. She received her Bacherlor's degree from Gonzaga University in Biology. Her research focuses on the development of cellulose-derived biofuel. As a member of the USU iGEM team, she was involved in the development and design of the project, as well as participation in the laboratory procedures. She also contributed to the development of the wiki by providing an abstract of the project.<br />
<br />
*'''Trent Mortensen''' is a finishing senior in Biological Engineering. His main research direction is biomedical in nature, using herbal antibiotics as possible alternatives for disease treatment. As part of the USU iGEM team, he played an active role in the planning of the project, group coordination, advising professor consultation, laboratory work, ordering of materials, lab maintenance, Wiki preparation, documentation, and presentation preparations throughout the course of the project.<br />
<br />
*'''Elisabeth Linton''' is a Biological Engineering student. Her research is focused on the the analysis and verification of gas chromatography and nuclear magnetic resonance methods for the quantification of intracellular polyhydroxyalkanoates in bacterial and environmental samples. For the iGEM team, Libbie helped in project determination and planning, as well as literature review and topic research. She also carried out work in the laboratory like restriction enzyme digestions, gel electrophoresis, DNA isolation, and bacterial transformations. She was also responsible for coordinating efforts and organizing the wiki.<br />
<br />
*'''Daniel Nelson''' is pursuing a degree in Biological Engineering and his current personal research project is “Omega-3 Fatty Acid Production and Extraction in Schizochytrium limacinum SR21.” He has enjoyed participating in group discussions as project ideas and direction have been determined. In the lab, he has worked with the team to isolate, purify, and analyze the key DNA promoters for the PHB synthesis gene. He hopes that through study of these promoter regions, the team will find a way to increase PHB production and create a general gene expression system to monitor cellular product accumulation. Outside of the lab, he has helped to design the logo for the USU team.<br />
<br />
*'''Rachel Porter''' is a sophomore at Utah State University majoring in Biological Engineering/pre-med. For the iGEM project, she helped to carry out some of the lab work. In the lab, she helped isolate and purify DNA, prepare and run electrophoresis gels, and culture cells.<br />
<br />
*'''Matthew Sims''' is a Senior attending Logan High School. This is his first year participating in laboratory research at Utah State University. He plans to go to college next fall and continue his studies in molecular biology and biochemistry. He helped during the summer months to carry out many experiments and obtain and analyze data.<br />
<br />
*'''Garrett Hinton''' is a junior at Sky View High School in Smithfield, Utah. He was heavily involved in the project discussion meetings, as well as the background literature research. Garrett spent many hours working on this project and carried out a substantial portion of the laboratory work.<br />
<br />
'''''We would like to thank our faculty advisers - Dr. Ron Sims, Dean Scott Hinton, and Dr. Charlie Miller - for all of their support and for providing this opportunity for the students.'''''<br />
[[Image:Advisors.jpg|350px|center]]<br />
<br />
== '''Logan and USU''' ==<br />
<html><br />
<style type="text/css"><br />
<br />
</style> <br />
<b><a href="http://www.usu.edu/">Utah State University</a></b> is located in Logan, Utah. Logan is about 85 miles north of Salt Lake City, Utah. The city of Logan is located in the heart of Cache Valley near the on the western slopes of the Bear River Mountains, the northernmost branch of the Wasatch Range. The city has a population of approximately 47,000. Logan was established in 1859 and has a rich heritage and wonderful culture. The city of Logan has been stated to be among the safest cities in America.<br><br />
<br><br />
Utah State University was established in 1888 as the Agricultural College of Utah. It's name was later changed to Utah State Agricultural College and was again changed to Utah State University (USU) in 1957. As the land-grant university in Utah, USU conducts world-class research in a variety of agricultural and natural resource disciplines, and has several projects in conjunction with the Department of Defense, NASA. Utah State University also conducts extensive aerospace research. The main campus is located in Logan, Utah. Beyond the Logan campus, Utah State's Extension programs extend academic resources and support throughout the entire state of Utah, having extension locations in each of Utah's 29 counties.<br><br />
<br />
<br />
</html><br />
[[Image:Sant_Blgd.jpg|600px|center]]</div>Liblinthttp://2008.igem.org/Team:Utah_State/TeamTeam:Utah State/Team2008-10-30T03:31:38Z<p>Liblint: /* iGEM 2008 at USU */</p>
<hr />
<div>{| align="center"<br />
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|}<br><br />
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#99CCFF">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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== '''iGEM 2008 at USU''' ==<br />
[[Image:USU_iGEM.jpg|500px|right]]<br />
|'''TEAM USU BACKGROUND'''<br />
Utah State University is proud to be involved in the 2008 iGEM competition for its first year. The 2008 USU iGEM team consists of graduate, undergraduate, and high school students with varying levels of experience in genetic and biological engineering under the supervision of faculty with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Though many project topics were seriously discussed, the team chose to study a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction.<br />
|-|[[Image:USU_iGEM.jpg|right|frame]]<br />
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<br />
This project was carried out at Utah State University in beautiful Logan, Utah.<br />
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The Utah State University iGEM team project is focused on<br />
creating an efficient system for production and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in Cupriavidus necator. In order to develop an<br />
optimal PHB detection system, we focused on the identification of the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow our reporter to indicate when PHB production was maximized.<br />
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== '''Team Members''' ==<br />
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'''FACULTY ADVISORS:'''<br />
*'''Scott Hinton''': Dean of the College of Engineering, USU<br />
*'''Dr. Charles Miller''': Department of Biological and Irrigation Engineering, USU<br />
*'''Dr. Ronald C. Sims''': Department of Biological and Irrigation Engineering, USU<br />
<br />
'''GRADUATE STUDENTS:''' <br />
*'''Junling Huo''': PhD student, Department of Biological and Irrigation Engineering, USU<br />
*'''Steven Merrigan''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Joseph Camire''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Kirsten Sims''': MS student, Department of Biological and Irrigation Engineering, USU <br />
<br />
'''UNDERGRADUATE STUDENTS:'''<br />
*'''Trent Mortensen''': "iGem spelled b-i-e, iGem" <br />
*'''Elisabeth Linton''': Department of Biological and Irrigation Engineering, USU <br />
*'''Daniel Nelson''': Department of Biological and Irrigation Engineering, USU <br />
*'''Rachel Porter''': Department of Biological and Irrigation Engineering, USU <br />
<br />
'''HIGH SCHOOL STUDENTS:'''<br />
*'''Garrett Hinton''': Sky View High School<br />
*'''Matthew Sims''': Logan High School<br />
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<gallery><br />
Image:Scott_Hinton.jpg|Dean Scott Hinton<br />
Image:Charles-Miller.jpg|Dr. Charlie Miller<br />
Image:Ron_sims.jpg|Dr. Ron Sims<br />
Image:JH.jpg|Junlng Huo<br />
Image:JC.jpg|Joseph Camire<br />
Image:Stephen_Merrigan.JPG|Stephen Merrigan<br />
Image:Libby_linton.jpg|Elisabeth Linton<br />
Image:DN.jpg|Daniel Nelson<br />
Image:rachel_249(2).jpg|Rachel Porter<br />
Image:TM.jpg|Trent Mortensen<br />
Image:Sims.jpg|Kirsten and Matthew Sims<br />
Image:GH.jpg|Garrett Hinton<br />
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== '''Team Member Contributions''' ==<br />
*'''Junling Huo''' is a PhD student in Biological Engineering who served as an advisor for the iGEM team. He helped to answer student questions, and guide such experimental activities as primer design, electrophoresis, and DNA purification. His personal research is on ''Rhodobacter sphaeroides'', a photosynthetic bacterium.<br />
<br />
*'''Stephen Merrigan''' is currently working on a MS in Biological Engineering at Utah State studying biodiesel production from algae, and has a background in Microbiology. Stephen's primary role was as an advisor for the project. During the first few months of the project Stephen did background research on project topics. He also helped with lab work when necessary, including DNA purification. Stephen also advised on the wiki construction.<br />
<br />
*'''Joseph Camire''' advised on targeting the amplification of the full phaCAB cassette and phaC gene,optimizing the PCR process and testing of primer sets with and without prefix and suffix regions. His work also involved sequence determination and analysis of the phaCAB cassette contained in the source plasmid. He also guided work on the phaC gene, which included targeting at amplification and preparing the gene for site-directed mutagenesis for removal of a critical restriction enzyme site prior to ligation of a new phaCAB cassette for biobrick construction.<br />
<br />
*'''Kirsten Sims''' is a first-year graduate student at Utah State University in Biological and Irrigation Engineering. She received her Bacherlor's degree from Gonzaga University in Biology. Her research focuses on the development of cellulose-derived biofuel. As a member of the USU iGEM team, she was involved in the development and design of the project, as well as participation in the laboratory procedures. She also contributed to the development of the wiki by providing an abstract of the project.<br />
<br />
*'''Trent Mortensen''' is a finishing senior in Biological Engineering. His main research direction is biomedical in nature, using herbal antibiotics as possible alternatives for disease treatment. As part of the USU iGEM team, he played an active role in the planning of the project, group coordination, advising professor consultation, laboratory work, ordering of materials, lab maintenance, Wiki preparation, documentation, and presentation preparations throughout the course of the project.<br />
<br />
*'''Elisabeth Linton''' is a Biological Engineering student. Her research is focused on the the analysis and verification of gas chromatography and nuclear magnetic resonance methods for the quantification of intracellular polyhydroxyalkanoates in bacterial and environmental samples. For the iGEM team, Libbie helped in project determination and planning, as well as literature review and topic research. She also carried out work in the laboratory like restriction enzyme digestions, gel electrophoresis, DNA isolation, and bacterial transformations. She was also responsible for coordinating efforts and organizing the wiki.<br />
<br />
*'''Daniel Nelson''' is pursuing a degree in Biological Engineering and his current personal research project is “Omega-3 Fatty Acid Production and Extraction in Schizochytrium limacinum SR21.” He has enjoyed participating in group discussions as project ideas and direction have been determined. In the lab, he has worked with the team to isolate, purify, and analyze the key DNA promoters for the PHB synthesis gene. He hopes that through study of these promoter regions, the team will find a way to increase PHB production and create a general gene expression system to monitor cellular product accumulation. Outside of the lab, he has helped to design the logo for the USU team.<br />
<br />
*'''Rachel Porter''' is a sophomore at Utah State University majoring in Biological Engineering/pre-med. For the iGEM project, she helped to carry out some of the lab work. In the lab, she helped isolate and purify DNA, prepare and run electrophoresis gels, and culture cells.<br />
<br />
*'''Matthew Sims''' is a Senior attending Logan High School. This is his first year participating in laboratory research at Utah State University. He plans to go to college next fall and continue his studies in molecular biology and biochemistry. He helped during the summer months to carry out many experiments and obtain and analyze data.<br />
<br />
*'''Garrett Hinton''' is a junior at Sky View High School in Smithfield, Utah. He was heavily involved in the project discussion meetings, as well as the background literature research. Garrett spent many hours working on this project and carried out a substantial portion of the laboratory work.<br />
<br />
'''''We would like to thank our faculty advisers - Dr. Ron Sims, Dean Scott Hinton, and Dr. Charlie Miller - for all of their support and for providing this opportunity for the students.'''''<br />
[[Image:Advisors.jpg|350px|center]]<br />
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== '''Logan and USU''' ==<br />
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<b><a href="http://www.usu.edu/">Utah State University</a></b> is located in Logan, Utah. Logan is about 85 miles north of Salt Lake City, Utah. The city of Logan is located in the heart of Cache Valley near the on the western slopes of the Bear River Mountains, the northernmost branch of the Wasatch Range. The city has a population of approximately 47,000. Logan was established in 1859 and has a rich heritage and wonderful culture. The city of Logan has been stated to be among the safest cities in America.<br><br />
<br><br />
Utah State University was established in 1888 as the Agricultural College of Utah. It's name was later changed to Utah State Agricultural College and was again changed to Utah State University (USU) in 1957. As the land-grant university in Utah, USU conducts world-class research in a variety of agricultural and natural resource disciplines, and has several projects in conjunction with the Department of Defense, NASA. Utah State University also conducts extensive aerospace research. The main campus is located in Logan, Utah. Beyond the Logan campus, Utah State's Extension programs extend academic resources and support throughout the entire state of Utah, having extension locations in each of Utah's 29 counties.<br><br />
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[[Image:Sant_Blgd.jpg|600px|center]]</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProtocolsTeam:Utah State/Protocols2008-10-30T03:30:55Z<p>Liblint: /* Streak Plates and Liquid Cultures from Transformed Colonies */</p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#99CCFF">Protocols</font>]]<br />
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=== Bacterial Transformation === <br />
<br />
[[Image:TrentLab1.jpg|300px|right]]<br />
Once the target DNA has been successfully ligated into the plasmid vector, the plasmid must be transferred into the host cell for replication and cloning. In order to do this, the bacterial cells must first be made “competent.” The term “competent” is to describe a cell state in which there exist gaps or openings in the cell wall which will allow the plasmid containing the target genes to enter into the cell. Several methods to make bacterial cells competent exist, such as the calcium chloride method and electroporation. Competent cells may also be purchased commercially. The team at USU has purchased competent cells for all experiments. The following is the method used by the USU team to insert the plasmids containing the PHB biobricks into the cells. <br />
''Trent Mortensen is shown in the right image inspecting agar plates containing transforming cells''<br />
<br />
'''Method'''<br><br />
1. Ensure the necessary antibiotic agar plates have been prepared or begin their preparation now. Four plates per transformation will be necessary (two today, then two tomorrow for streaking). Also ensure that 10 ml liquid media is made up per transformation (also for tomorrow).<br><br />
2. Clean punchout tool by dipping in 10% chlorox, deionized water, deionized water again, then 80%aq ethanol. Let dry for 2 minutes and repeat cleaning procedure between punchouts. <br><br />
3. Punch out gene of choice with a twisting motion, allowing the metal to cut the paper. Use the center part of the punchout tool to dislodge the paper into a 2.5 ml microcentrifuge tube. <br> <br />
4. Add 5 μl TE buffer, place in a 42˚C water bath, and allow plasmids in the paper to elute for 20 minutes.<br />
5. Centrifuge tube(s) at 15K RPM for 3 minutes. Remove SOC media from the -20˚C freezer and leave out to thaw.<br><br />
6. Take competent cells from the -80˚C freezer and place on an ice bath.<br><br />
7. Pipette contents of tube up and down a few times then take 2 μl of the DNA solution and add to the competent cells. Ensure the pipetting is done directly into the cell solution. Let cells incubate in the ice bath for 30 minutes. Heat water bath to 42˚C.<br><br />
8. Heat shock cells in the 42˚C water bath for 30 seconds. Remove and place back in the ice bath for 2 minutes.<br><br />
9. In the hood, add 250 μl SOC media to each tube, bringing the total cell solution to 300 μl. Incubate at 37˚C for 1 hour.<br><br />
10. Get out the antibiotic agar plates. In the hood, add 200 μl of each transformed cell solution to the appropriate antibiotic plate. Use the Bunsen burner to create a “hockey stick” out of a glass pipette tip by holding over the flame until it bends. Allow to cool. Spread cell solution uniformly over the agar plate using the “hockey stick,” then before discarding, spread residual solution on the “stick” over a second plate to get more a more sparse colony distribution. <br><br />
11. Parafilm all plates and place in 37˚C incubator 12-14 hours, or overnight if that is not possible. <br><br />
<br />
=== Streak Plates and Liquid Cultures from Transformed Colonies ===<br />
<br />
After bacterial cells have been transformed, successfully transformed cells must be selected. Because 100% of the cells do not receive the desired plasmid and target gene, it is essential to select for cells that do have the target genes. Several methods are used to accomplish this, such as incorporation of antibiotic resistance and also the lac operon. The USU team has used antibiotic resistance to select for successful transformations. To do this, an antibiotic resistance gene is also added to the plasmid vector that contains the target genes. By doing so, it is possible to know that a cell was successfully transformed based on its ability to grow on an agar plate with antibiotics added. Because the cell is able to grow, the antibiotic resistance gene must be present as well as the target gene. From the agar plates containing the antibiotics, a colony is picked off and transferred into a liquid culture for further analysis and cellular production. The following is the method used by USU to clone the DNA and select for the successful transformation of the PHB biobrick into the cells.<br />
<br />
'''Method'''<br><br />
1. Prepare two 12 ml tubes per transformation, each with 5 ml media containing the appropriate antibiotic (if felt necessary). Get out antibiotic agar plates. Inspect plates from yesterday for colonies. At least two colonies are preferred, but one will do. Select two colonies and label them. <br><br />
2. Use a pipette tip to extract half of each colony and inoculate one agar plate per colony. Using a pipette with a tip, extract the other half of each colony and inoculate one liquid media tube per colony. Label all tubes and plates and place in the 37˚C incubator until tomorrow morning. <br><br />
<br />
=== Preparation for DNA Separation ===<br />
[[Image:Microcentrifuge.JPG|300px|right]]<br />
Following successful bacterial cloning and isolation, it is important to verify that the target gene is in the cell and that the plasmid is functional. To do this, it is a common practice to sequence the DNA plasmid. To obtain enough DNA for sequencing, the bacterial clones are grown in a liquid culture to allow for plasmid replication within the cell and also large amounts of biomass containing these plasmids. The cells are harvested by centrifugation and then prepared for DNA plasmid extraction. DNA plasmid extraction can be done several ways, and the overall purpose is to lyse the cells and separate the plasmid DNA from all other cellular proteins, DNA, and debris. The following is the method used by the USU team to isolate the plasmid DNA containing the PHB biobricks. ''An Eppendorf Microcentrifuge was used in these experiments and is shown on the right.''<br />
<br />
'''Method'''<br><br />
1. Prepare two water baths, one boiling and the other 68˚C. <br> <br />
2. Centrifuge the 12 ml tubes containing the 5 ml cultures in the large centrifuge at 8K RPM for 20 min. Discard supernatant. <br> <br />
3. Resuspend cells in 200 μl of STET buffer. <br> <br />
4. Add 10 μl (if older preparation) Lysozyme (50 mg/ml) and incubate at room temperature for 5 min. <br> <br />
5. Boil for 45 sec and centrifuge for 20 min at 13K RPM (or until pellet gets tight).<br> <br />
6. Use a pipette tip to remove the pellet.<br> <br />
7. Add 5 μl RNase A (10 mg/ml) and incubate at 68˚C for 10 minutes.<br> <br />
8. Add 10 μl of 5% CTAB and incubate at room temperature for 3 min.<br> <br />
9. Centrifuge for 5 min at 13K RPM, discard supernatant, and resuspend in 300 μl of 1.2 M NaCl using a vortex mixer.<br> <br />
10. Add 750 μl of ethanol and centrifuge for 5 min at 13K RPM.<br> <br />
11. Discard supernatant, rinse pellet (cannot see) in 80% ethanol, and let tubes dry upside down with caps open.<br><br />
<br />
=== Restriction Enzyme Digestion and Electrophoresis ===<br />
<br />
Restriction enzyme digestion is the process by which a target DNA sequence is separated from the rest of the DNA molecule. Specific knowledge of the target DNA gene is needed to determine which enzyme and conditions to use during the digestion reaction. Once the target DNA sequence is known and the correct enzymes have been selected, the DNA may be digested. Listed below is the procedure used by USU to digest the cellular DNA and obtain the target DNA of PHB synthesis and gene promotion. After enzyme digestion, electrophoresis is used to separate the plasmid DNA from the target DNA genes. A gel is prepared and the respective reaction mixes are loaded into the gel. Using a DNA ladder, and knowing the size of the target DNA gene, the corresponding band can be seen and cut out of the gel. The target DNA may then be removed and isolated from the gel, thus yielding the desired target DNA genes. The DNA from this may then be used in PCR reactions, sequencing, ligations for further experimentation, and many more. Listed below are the protocols used by the USU team to run the electrophoresis reaction. <br />
<br />
'''Method'''<br><br />
1. Resuspend DNA in 40 μl water, vortex, and do a brief centrifuge to get solution to the bottom of the tube. <br><br />
2. Add components to the digestion solution in the following order: DNA (23 μl), 10X Tango buffer (3 μl), Xba1 (2 μl), and Pst1 (2 μl). The volume and restriction enzymes can be varied, but it should be ensured that the total volume is 10X the amount of Tango buffer added. Tap tubes periodically and allow to digest while preparing electrophoresis gel. <br><br />
3. Prepare electrophoresis gel by adding 2 g agarose to 200 ml TAE (1% solution). This is best done in an Erlenmeyer flask of adequate volume as swirling will need to be done. Place in the microwave and microwave on high for 20 seconds at a time, pulling it out and swirling until solution is homogeneous again, then repeating (BE CAREFUL to watch the solution closely when swirling – it superheats and can boil over and cause severe burns). Continue until solution is seen boiling in the microwave then gently swirl again. <br><br />
4. Add 20 μl ethidium bromide to solution and swirl until dissolved evenly. <br><br />
5. Add 6 μl of 6X loading dye to each tube of digested DNA solution. <br><br />
6. Prepare the electrophoresis unit by orienting the basin sideways with rubber gaskets firmly against the side. Place desired well template in the basin. <br><br />
7. When the agarose solution is cool enough to comfortably touch the flask, pour into the basin until the solution is about ¾ of the way to the top of the well template. <br><br />
8. When the gel is solidified (should look somewhat cloudy), remove the well template and change basin orientation to have the wells closest to the negative pole (as the DNA will flow towards the positive pole). Pour 1X TAE buffer into both sides of the electrophoresis unit until it just covers the gel and fills the wells.<br><br />
9. By inserting the pipette tip below the TAE liquid and into the well, add 10 μl of DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the digested DNA solutions to the wells by adding about 2 μl less than the total volume in the tubes to prevent air bubbles in the wells.<br><br />
10. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 70 V (this can be as high as 100 V if time is an issue), and press the start button. Separation should take two to three hours. The yellow dye shows the location of the smaller nucleotide lengths and the blue dye shows the location of the larger nucleotide lengths. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. The basin can then be placed back in the electrophoresis unit for further separation if desired. Take care to not have the power supply on without the lid to the unit in place. <br><br />
11. When the desired level of separation is attained, the basin can be placed on the transilluminator for picture taking. Place the cone-shaped cover over the transilluminator and place the digital camera in the top hole for pictures.<br><br />
<br />
''The electrophoresis units and UV transilluminator used in this project are shown in the left and center images below. An electrophoresis image is given on the right.''<br />
<br />
[[Image:Electrophoresis_Units.JPG|320px]][[Image:UV_Transilluminator.JPG|320px]][[Image:Elect.jpg|320px]]<br />
<br />
=== Media Preparation ===<br />
<br />
For all experimentation involving the need for bacterial biomass and experimentation, proper media is needed to grow the cells. The following is the media composition and methods used by USU to prepare the media.<br />
<br />
1. Add 5 g yeast extract, 10 g NaCl, 10 g Bacto Tryptone, and 15 g agar (if desired) to a 2 L Erlenmeyer flask and bring the volume up to 1 L with ddH20. Mix by swirling. Cover top with foil.<br><br />
2. Autoclave for 45 minutes (liquid setting, 0 minutes drying time). It will take an additional half hour for the autoclave to finish cooling then an additional 20 to 30 minutes until the media is cool enough to pour.<br><br />
<br />
=== 1X TAE Preparation ===<br />
1. Add 40 ml 50X TAE solution to a 2 L flask and bring level up to 2 L with ddH20. <br><br />
<br />
=== Polymerase Chain Reaction (PCR) ===<br />
[[Image:Thermocycler.JPG|250px|right]]<br />
<br />
When large amounts of DNA are needed it is necessary to do a PCR reaction. When a PCR reaction is set up and operated properly it will amplify a single target sequence of DNA. The reaction is first set up by designing primers to use that will bind only to the desired regions of the DNA sequence to be replicates and choosing a polymerase to copy the DNA. Once the primer and polymerase has been chosen, the reaction conditions of time and temperature must be optimized. Thus, when the reaction works properly only the target DNA will be amplified into large quantities that may then be isolated and used for further experimentation. The following is the procedure used by USU for PCR reactions to amplify the PHB synthesis and promotion genes.<br> ''An Eppendorf Thermocycle was used in these experiments, as shown in the image on the right.''<br />
<br />
'''Method'''<br><br />
1. Obtain the following reagents from the freezer: DNA template (cells or DNA), 10X Taq buffer (+KCl, -Mg/Cl<small>2</small>), MgCl<small>2</small>, 10 mM dNTP Mix, Taq polymerase (take out of freezer only immediately when needed and put back), and sterile distilled H<small>2</small>O. Place all reagents on ice. Also obtain PCR (either 0.2 or 0.5 ml) tubes.<br><br />
2. Add the following reagents to a tube (50 μl reaction) in the following volumes and order:<br><br />
• 32 μl sterile H<small>2</small>O,<br><br />
• 5 μl 10X buffer, <br><br />
• 2 μl dNTP Mix, <br><br />
• 6 μl MgCl<small>2</small><br><br />
• 3 μl cells/DNA, <br><br />
• 0.25 μl Taq Polymerase<br><br />
• 1 μl primer 1<br><br />
• 1 μl primer 2<br><br />
MgCl<small>2</small> volume can be varied (lower to increase specificity – just ensure total volume is 50 ul with H<small>2</small>O). If many reactions are to be constructed, a master mix can be made up to cut down on time and pipette tip usage (if this is done, ensure primers are added to the appropriate reaction, i.e. perhaps not to the master mix). Tap or vortex tubes and take to the thermocyler. Place all reagents back in the -20˚C freezer. <br><br />
3. Choose thermocycler temperatures. The Eppendorf Mastercycler will cycle between three temperatures: typical temperatures are 94˚C for denaturing, 50-60˚C for primer annealing, and 72˚C for polymerase extending. Lowering the annealing temperature decreases DNA specificity; 55˚C is a good temperature to begin if no trials have been made with the sample. <br><br />
4. Turn on thermocycler with the switch in the back of the unit and open the lid. The placement of the tubes depends on the size of the tube (0.2 or 0.5 ml) and whether or not a temperature gradient is to be used. <br><br><br />
<br />
* If no gradient will be used, tubes can be placed anywhere on the unit in the appropriately-sized hole. Select “Files” and press enter. Select “Load” and then “Standard.” If cells will be used in the reaction, include a 1-minute lysing step at the beginning (step 1); this will be followed by a 1-minute DNA denaturing step (step 2). If purified DNA will be used, set step 1 to 1 second. Set an annealing temperature for step 3. Ensure the lid temperature is 105˚C and the extending temperature is 72˚C. Press exit. If prompted to save, save by pressing enter three times. Press exit to return to the main menu. Choose “Start” on the main menu and select “Standard.” The program should begin. <br><br />
<br />
* If a gradient is to be used, temperature will vary according to column. A 20˚C range is the maximum range that can be used (+/- 10˚C). The range is made by setting a temperature for the middle column and then setting a +/- range. To see what the temperatures will be if a gradient is used, select “OPTIONS” on the main menu, then select “Gradient.” Select the size tube that is being used by pressing “Sel,” then press enter. Choose a temperature for the center column, press enter, then select a +/- range and press enter. The column number along with the corresponding temperature is shown. Decide tube placement based on this information. Press exit twice to return on the main menu. Select “Files” then “Load,” then “Gradient.” If cells are being used, set the cell lysing step (step 1) to 1 minute (1:00); if purified DNA is being used, set this time to 1 second (0:01). Step 2 should be 94C, Step 4 should be 72˚C, and the lid temperature should be 105˚C. Go to step 3 and set an annealing temperature for the center column. Leave the next two lines as they are, and change the gradient setting (“G”) to the +/- the center temperature amount. Press exit. If prompted to save, press enter three times; if not prompted to save, press enter once. Press exit to get back to the main menu. To begin cycle, select “Start,” then select “Gradient.” The program should begin. <br><br />
<br />
5. The thermocycler is set to store the completed reaction tubes at 4˚C when finished. <br><br />
<br />
=== Ligation ===<br />
<br />
Ligation is the process by which the insert (target DNA gene) is inserted into a plasmid. Both the plasmid and insert have been digested and have the proper “sticky” or blunt ends which are compatible for joining the two DNA pieces together into one molecule. These two DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid. This newly formed plasmid may then be isolated using gel electrophoresis and then used for bacterial transformation or other experimentation. The following is the procedure used by USU to ligate PHB genes into the biobrick plasmids. <br><br />
'''Method'''<br><br />
1. Obtain the following reagents, some of which are in the -20˚C freezer: DNA vector, DNA insert, 10X ligation buffer, T4 DNA ligase (take out only when needed, then return immediately to freezer), and sterile distilled water.<br><br />
2. Ideally, it is desirable to have the concentration of insert ends (or moles of insert) be two to three times the concentration of vector ends (or moles of vector), with a total DNA concentration of 50-400 ng/μl in the reaction. If determining the DNA concentration is not possible, place two to three times the volume of vector as the volume of insert in the reaction. As this is often the case, place the following reagents in a thin-walled PCR tube in the following volumes:<br><br />
<br />
• 10 μl insert DNA<br><br />
• 3 μl vector DNA<br><br />
• 2 μl 10X ligation buffer<br><br />
• 4 μl H2O<br><br />
• 1 μl T4 DNA ligase<br><br />
= ''20 μl total''<br><br />
<br />
This could also be done in different volumes depending on DNA concentration/total volume desired.<br><br />
3. Gently mix the tube, and place the tube in the PCR thermocyler, turn on the machine, select “Start,” from the main menu, select “22” and press “Start.” The thermocycler will keep the reaction at 22˚C.<br><br />
4. Incubate for 60 minutes. Heat-inactivate by placing tubes in 68C water bath for 10 minutes. Place in the freezer if storing for later use. <br></div>Liblinthttp://2008.igem.org/Team:Utah_State/TeamTeam:Utah State/Team2008-10-30T03:30:24Z<p>Liblint: /* iGEM 2008 at USU */</p>
<hr />
<div>{| align="center"<br />
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|}<br><br />
{|style="font color="#CC3300"; background-color:#212223; cellpadding="3" cellspacing="5" border="2" bordercolor="#cd0000"border-spacing:6px; text-align:center" width="960px"<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State|<font color="#ffffff">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#99CCFF">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
<br />
<html><br />
<style type="text/css"><br />
body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
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</style> <br />
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<br />
{|align="justify"<br />
== '''iGEM 2008 at USU''' ==<br />
[[Image:USU_iGEM.jpg|500px|right]]<br />
|'''TEAM USU BACKGROUND'''<br />
Utah State University is proud to be involved in the 2008 iGEM competition for its first year. The 2008 USU iGEM team consists of graduate, undergraduate, and high school students with varying levels of experience in genetic and biological engineering under the supervision of faculty with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Through team meetings and working closely together in the lab, team members came to know each other and the project material. Though many project topics were seriously discussed, the team chose to study a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction.<br />
|-|[[Image:USU_iGEM.jpg|right|frame]]<br />
<br />
<br />
This project was carried out at Utah State University in beautiful Logan, Utah.<br />
<br />
|-<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for production and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in Cupriavidus necator. In order to develop an<br />
optimal PHB detection system, we focused on the identification of the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow our reporter to indicate when PHB production was maximized.<br />
|<br />
|-<br />
|<br />
|<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|border = "10"<br />
|-<br />
|rowspan="3"|<br />
'''FACULTY ADVISORS:'''<br />
*'''Scott Hinton''': Dean of the College of Engineering, USU<br />
*'''Dr. Charles Miller''': Department of Biological and Irrigation Engineering, USU<br />
*'''Dr. Ronald C. Sims''': Department of Biological and Irrigation Engineering, USU<br />
<br />
'''GRADUATE STUDENTS:''' <br />
*'''Junling Huo''': PhD student, Department of Biological and Irrigation Engineering, USU<br />
*'''Steven Merrigan''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Joseph Camire''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Kirsten Sims''': MS student, Department of Biological and Irrigation Engineering, USU <br />
<br />
'''UNDERGRADUATE STUDENTS:'''<br />
*'''Trent Mortensen''': "iGem spelled b-i-e, iGem" <br />
*'''Elisabeth Linton''': Department of Biological and Irrigation Engineering, USU <br />
*'''Daniel Nelson''': Department of Biological and Irrigation Engineering, USU <br />
*'''Rachel Porter''': Department of Biological and Irrigation Engineering, USU <br />
<br />
'''HIGH SCHOOL STUDENTS:'''<br />
*'''Garrett Hinton''': Sky View High School<br />
*'''Matthew Sims''': Logan High School<br />
|<br />
<gallery><br />
Image:Scott_Hinton.jpg|Dean Scott Hinton<br />
Image:Charles-Miller.jpg|Dr. Charlie Miller<br />
Image:Ron_sims.jpg|Dr. Ron Sims<br />
Image:JH.jpg|Junlng Huo<br />
Image:JC.jpg|Joseph Camire<br />
Image:Stephen_Merrigan.JPG|Stephen Merrigan<br />
Image:Libby_linton.jpg|Elisabeth Linton<br />
Image:DN.jpg|Daniel Nelson<br />
Image:rachel_249(2).jpg|Rachel Porter<br />
Image:TM.jpg|Trent Mortensen<br />
Image:Sims.jpg|Kirsten and Matthew Sims<br />
Image:GH.jpg|Garrett Hinton<br />
</gallery><br />
|}<br />
<br />
== '''Team Member Contributions''' ==<br />
*'''Junling Huo''' is a PhD student in Biological Engineering who served as an advisor for the iGEM team. He helped to answer student questions, and guide such experimental activities as primer design, electrophoresis, and DNA purification. His personal research is on ''Rhodobacter sphaeroides'', a photosynthetic bacterium.<br />
<br />
*'''Stephen Merrigan''' is currently working on a MS in Biological Engineering at Utah State studying biodiesel production from algae, and has a background in Microbiology. Stephen's primary role was as an advisor for the project. During the first few months of the project Stephen did background research on project topics. He also helped with lab work when necessary, including DNA purification. Stephen also advised on the wiki construction.<br />
<br />
*'''Joseph Camire''' advised on targeting the amplification of the full phaCAB cassette and phaC gene,optimizing the PCR process and testing of primer sets with and without prefix and suffix regions. His work also involved sequence determination and analysis of the phaCAB cassette contained in the source plasmid. He also guided work on the phaC gene, which included targeting at amplification and preparing the gene for site-directed mutagenesis for removal of a critical restriction enzyme site prior to ligation of a new phaCAB cassette for biobrick construction.<br />
<br />
*'''Kirsten Sims''' is a first-year graduate student at Utah State University in Biological and Irrigation Engineering. She received her Bacherlor's degree from Gonzaga University in Biology. Her research focuses on the development of cellulose-derived biofuel. As a member of the USU iGEM team, she was involved in the development and design of the project, as well as participation in the laboratory procedures. She also contributed to the development of the wiki by providing an abstract of the project.<br />
<br />
*'''Trent Mortensen''' is a finishing senior in Biological Engineering. His main research direction is biomedical in nature, using herbal antibiotics as possible alternatives for disease treatment. As part of the USU iGEM team, he played an active role in the planning of the project, group coordination, advising professor consultation, laboratory work, ordering of materials, lab maintenance, Wiki preparation, documentation, and presentation preparations throughout the course of the project.<br />
<br />
*'''Elisabeth Linton''' is a Biological Engineering student. Her research is focused on the the analysis and verification of gas chromatography and nuclear magnetic resonance methods for the quantification of intracellular polyhydroxyalkanoates in bacterial and environmental samples. For the iGEM team, Libbie helped in project determination and planning, as well as literature review and topic research. She also carried out work in the laboratory like restriction enzyme digestions, gel electrophoresis, DNA isolation, and bacterial transformations. She was also responsible for coordinating efforts and organizing the wiki.<br />
<br />
*'''Daniel Nelson''' is pursuing a degree in Biological Engineering and his current personal research project is “Omega-3 Fatty Acid Production and Extraction in Schizochytrium limacinum SR21.” He has enjoyed participating in group discussions as project ideas and direction have been determined. In the lab, he has worked with the team to isolate, purify, and analyze the key DNA promoters for the PHB synthesis gene. He hopes that through study of these promoter regions, the team will find a way to increase PHB production and create a general gene expression system to monitor cellular product accumulation. Outside of the lab, he has helped to design the logo for the USU team.<br />
<br />
*'''Rachel Porter''' is a sophomore at Utah State University majoring in Biological Engineering/pre-med. For the iGEM project, she helped to carry out some of the lab work. In the lab, she helped isolate and purify DNA, prepare and run electrophoresis gels, and culture cells.<br />
<br />
*'''Matthew Sims''' is a Senior attending Logan High School. This is his first year participating in laboratory research at Utah State University. He plans to go to college next fall and continue his studies in molecular biology and biochemistry. He helped during the summer months to carry out many experiments and obtain and analyze data.<br />
<br />
*'''Garrett Hinton''' is a junior at Sky View High School in Smithfield, Utah. He was heavily involved in the project discussion meetings, as well as the background literature research. Garrett spent many hours working on this project and carried out a substantial portion of the laboratory work.<br />
<br />
'''''We would like to thank our faculty advisers - Dr. Ron Sims, Dean Scott Hinton, and Dr. Charlie Miller - for all of their support and for providing this opportunity for the students.'''''<br />
[[Image:Advisors.jpg|350px|center]]<br />
<br />
== '''Logan and USU''' ==<br />
<html><br />
<style type="text/css"><br />
<br />
</style> <br />
<b><a href="http://www.usu.edu/">Utah State University</a></b> is located in Logan, Utah. Logan is about 85 miles north of Salt Lake City, Utah. The city of Logan is located in the heart of Cache Valley near the on the western slopes of the Bear River Mountains, the northernmost branch of the Wasatch Range. The city has a population of approximately 47,000. Logan was established in 1859 and has a rich heritage and wonderful culture. The city of Logan has been stated to be among the safest cities in America.<br><br />
<br><br />
Utah State University was established in 1888 as the Agricultural College of Utah. It's name was later changed to Utah State Agricultural College and was again changed to Utah State University (USU) in 1957. As the land-grant university in Utah, USU conducts world-class research in a variety of agricultural and natural resource disciplines, and has several projects in conjunction with the Department of Defense, NASA. Utah State University also conducts extensive aerospace research. The main campus is located in Logan, Utah. Beyond the Logan campus, Utah State's Extension programs extend academic resources and support throughout the entire state of Utah, having extension locations in each of Utah's 29 counties.<br><br />
<br />
<br />
</html><br />
[[Image:Sant_Blgd.jpg|600px|center]]</div>Liblinthttp://2008.igem.org/Team:Utah_State/TeamTeam:Utah State/Team2008-10-30T03:29:08Z<p>Liblint: /* iGEM 2008 at USU */</p>
<hr />
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|}<br><br />
{|style="font color="#CC3300"; background-color:#212223; cellpadding="3" cellspacing="5" border="2" bordercolor="#cd0000"border-spacing:6px; text-align:center" width="960px"<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State|<font color="#ffffff">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#99CCFF">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
<br />
<html><br />
<style type="text/css"><br />
body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
<br />
<br />
</style> <br />
</html><br />
<br />
{|align="justify"<br />
== '''iGEM 2008 at USU''' ==<br />
[[Image:USU_iGEM.jpg|500px|right]]<br />
|'''TEAM USU BACKGROUND'''<br />
Utah State University is proud to be involved in the 2008 iGEM competition for its first year. The 2008 USU iGEM team consists of graduate, undergraduate, and high school students with varying levels of experience in genetic and biological engineering under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Through team meetings and working closely together in the lab, team members came to know each other and the project material. Though many project topics were seriously discussed, the team chose to study a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction.<br />
|-|[[Image:USU_iGEM.jpg|right|frame]]<br />
<br />
<br />
This project was carried out at Utah State University in beautiful Logan, Utah.<br />
<br />
|-<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for production and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in Cupriavidus necator. In order to develop an<br />
optimal PHB detection system, we focused on the identification of the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow our reporter to indicate when PHB production was maximized.<br />
|<br />
|-<br />
|<br />
|<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|border = "10"<br />
|-<br />
|rowspan="3"|<br />
'''FACULTY ADVISORS:'''<br />
*'''Scott Hinton''': Dean of the College of Engineering, USU<br />
*'''Dr. Charles Miller''': Department of Biological and Irrigation Engineering, USU<br />
*'''Dr. Ronald C. Sims''': Department of Biological and Irrigation Engineering, USU<br />
<br />
'''GRADUATE STUDENTS:''' <br />
*'''Junling Huo''': PhD student, Department of Biological and Irrigation Engineering, USU<br />
*'''Steven Merrigan''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Joseph Camire''': MS student, Department of Biological and Irrigation Engineering, USU <br />
*'''Kirsten Sims''': MS student, Department of Biological and Irrigation Engineering, USU <br />
<br />
'''UNDERGRADUATE STUDENTS:'''<br />
*'''Trent Mortensen''': "iGem spelled b-i-e, iGem" <br />
*'''Elisabeth Linton''': Department of Biological and Irrigation Engineering, USU <br />
*'''Daniel Nelson''': Department of Biological and Irrigation Engineering, USU <br />
*'''Rachel Porter''': Department of Biological and Irrigation Engineering, USU <br />
<br />
'''HIGH SCHOOL STUDENTS:'''<br />
*'''Garrett Hinton''': Sky View High School<br />
*'''Matthew Sims''': Logan High School<br />
|<br />
<gallery><br />
Image:Scott_Hinton.jpg|Dean Scott Hinton<br />
Image:Charles-Miller.jpg|Dr. Charlie Miller<br />
Image:Ron_sims.jpg|Dr. Ron Sims<br />
Image:JH.jpg|Junlng Huo<br />
Image:JC.jpg|Joseph Camire<br />
Image:Stephen_Merrigan.JPG|Stephen Merrigan<br />
Image:Libby_linton.jpg|Elisabeth Linton<br />
Image:DN.jpg|Daniel Nelson<br />
Image:rachel_249(2).jpg|Rachel Porter<br />
Image:TM.jpg|Trent Mortensen<br />
Image:Sims.jpg|Kirsten and Matthew Sims<br />
Image:GH.jpg|Garrett Hinton<br />
</gallery><br />
|}<br />
<br />
== '''Team Member Contributions''' ==<br />
*'''Junling Huo''' is a PhD student in Biological Engineering who served as an advisor for the iGEM team. He helped to answer student questions, and guide such experimental activities as primer design, electrophoresis, and DNA purification. His personal research is on ''Rhodobacter sphaeroides'', a photosynthetic bacterium.<br />
<br />
*'''Stephen Merrigan''' is currently working on a MS in Biological Engineering at Utah State studying biodiesel production from algae, and has a background in Microbiology. Stephen's primary role was as an advisor for the project. During the first few months of the project Stephen did background research on project topics. He also helped with lab work when necessary, including DNA purification. Stephen also advised on the wiki construction.<br />
<br />
*'''Joseph Camire''' advised on targeting the amplification of the full phaCAB cassette and phaC gene,optimizing the PCR process and testing of primer sets with and without prefix and suffix regions. His work also involved sequence determination and analysis of the phaCAB cassette contained in the source plasmid. He also guided work on the phaC gene, which included targeting at amplification and preparing the gene for site-directed mutagenesis for removal of a critical restriction enzyme site prior to ligation of a new phaCAB cassette for biobrick construction.<br />
<br />
*'''Kirsten Sims''' is a first-year graduate student at Utah State University in Biological and Irrigation Engineering. She received her Bacherlor's degree from Gonzaga University in Biology. Her research focuses on the development of cellulose-derived biofuel. As a member of the USU iGEM team, she was involved in the development and design of the project, as well as participation in the laboratory procedures. She also contributed to the development of the wiki by providing an abstract of the project.<br />
<br />
*'''Trent Mortensen''' is a finishing senior in Biological Engineering. His main research direction is biomedical in nature, using herbal antibiotics as possible alternatives for disease treatment. As part of the USU iGEM team, he played an active role in the planning of the project, group coordination, advising professor consultation, laboratory work, ordering of materials, lab maintenance, Wiki preparation, documentation, and presentation preparations throughout the course of the project.<br />
<br />
*'''Elisabeth Linton''' is a Biological Engineering student. Her research is focused on the the analysis and verification of gas chromatography and nuclear magnetic resonance methods for the quantification of intracellular polyhydroxyalkanoates in bacterial and environmental samples. For the iGEM team, Libbie helped in project determination and planning, as well as literature review and topic research. She also carried out work in the laboratory like restriction enzyme digestions, gel electrophoresis, DNA isolation, and bacterial transformations. She was also responsible for coordinating efforts and organizing the wiki.<br />
<br />
*'''Daniel Nelson''' is pursuing a degree in Biological Engineering and his current personal research project is “Omega-3 Fatty Acid Production and Extraction in Schizochytrium limacinum SR21.” He has enjoyed participating in group discussions as project ideas and direction have been determined. In the lab, he has worked with the team to isolate, purify, and analyze the key DNA promoters for the PHB synthesis gene. He hopes that through study of these promoter regions, the team will find a way to increase PHB production and create a general gene expression system to monitor cellular product accumulation. Outside of the lab, he has helped to design the logo for the USU team.<br />
<br />
*'''Rachel Porter''' is a sophomore at Utah State University majoring in Biological Engineering/pre-med. For the iGEM project, she helped to carry out some of the lab work. In the lab, she helped isolate and purify DNA, prepare and run electrophoresis gels, and culture cells.<br />
<br />
*'''Matthew Sims''' is a Senior attending Logan High School. This is his first year participating in laboratory research at Utah State University. He plans to go to college next fall and continue his studies in molecular biology and biochemistry. He helped during the summer months to carry out many experiments and obtain and analyze data.<br />
<br />
*'''Garrett Hinton''' is a junior at Sky View High School in Smithfield, Utah. He was heavily involved in the project discussion meetings, as well as the background literature research. Garrett spent many hours working on this project and carried out a substantial portion of the laboratory work.<br />
<br />
'''''We would like to thank our faculty advisers - Dr. Ron Sims, Dean Scott Hinton, and Dr. Charlie Miller - for all of their support and for providing this opportunity for the students.'''''<br />
[[Image:Advisors.jpg|350px|center]]<br />
<br />
== '''Logan and USU''' ==<br />
<html><br />
<style type="text/css"><br />
<br />
</style> <br />
<b><a href="http://www.usu.edu/">Utah State University</a></b> is located in Logan, Utah. Logan is about 85 miles north of Salt Lake City, Utah. The city of Logan is located in the heart of Cache Valley near the on the western slopes of the Bear River Mountains, the northernmost branch of the Wasatch Range. The city has a population of approximately 47,000. Logan was established in 1859 and has a rich heritage and wonderful culture. The city of Logan has been stated to be among the safest cities in America.<br><br />
<br><br />
Utah State University was established in 1888 as the Agricultural College of Utah. It's name was later changed to Utah State Agricultural College and was again changed to Utah State University (USU) in 1957. As the land-grant university in Utah, USU conducts world-class research in a variety of agricultural and natural resource disciplines, and has several projects in conjunction with the Department of Defense, NASA. Utah State University also conducts extensive aerospace research. The main campus is located in Logan, Utah. Beyond the Logan campus, Utah State's Extension programs extend academic resources and support throughout the entire state of Utah, having extension locations in each of Utah's 29 counties.<br><br />
<br />
<br />
</html><br />
[[Image:Sant_Blgd.jpg|600px|center]]</div>Liblinthttp://2008.igem.org/Team:Utah_StateTeam:Utah State2008-10-30T03:28:29Z<p>Liblint: </p>
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!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Utah_State|<font color="#99CCFF">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#ffffff">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Notebook|<font color="#ffffff">Notebook</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Protocols|<font color="#ffffff">Protocols</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Links|<font color="#ffffff">Links</font>]]<br />
|}<br />
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<style type="text/css"><br />
body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
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{|align="justify"<br />
[[Image:IGEMBanner.jpg|960 px]]<br />
[[Image:Igemusu.jpg|350px|left|]]<br />
<br />
Utah State University is proud to be involved for the first time in the 2008 iGEM competition. The 2008 USU iGEM team consists of graduate and undergraduate students in Biological Engineering, as well as two high school students, working under the supervision of professors with backgrounds in Biological Engineering, Electrical Engineering, Biology, and Microbiology. Prior to this project, none of the undergraduate or high school students had experience with genetic engineering or synthetic biology. This project provided a great opportunity for these students to learn about synthetic biology and its potential, as well as to gain a great deal of hands on experience with laboratory methods. Through team meetings and working closely together in the lab, team members were also provided with opportunities to get to know each other and the specific project material. <br />
<br />
Though many project topics were seriously discussed, the team chose the project for developing a method of monitoring Polyhydroxybutyrate production in microorganisms by inserting a reporter in the PHB cassette. This project was selected because of its potential to make the PHB production process more efficient and cost effective by creating a simple system for determining the optimum time for PHB extraction. This project was carried out from May to October of 2008.<br />
<br />
<br><br />
USU is located in Logan, UT and is nestled in the beautiful mountains of Cache Valley. For those interested in a great education and are looking for amazing skiing and other outdoor adventures and recreation, USU is perfect!!<br />
[[Image:Utah.jpg|500px|left]][[Image:CacheValley.jpg|450px|right]]<br />
<br />
<br />
<br />
|}</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:27:38Z<p>Liblint: /* References */</p>
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|}<br><br />
{|style="font color="#CC3300"; background-color:#212223; cellpadding="3" cellspacing="5" border="2" bordercolor="#cd0000"border-spacing:6px; text-align:center" width="960px"<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State|<font color="#ffffff">Home</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Team|<font color="#ffffff">The Team</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Project|<font color="#99CCFF">The Project</font>]]<br />
!style="text-align:center; background-color:#212223; border-width:0px; padding:2px;"|[[Team:Utah_State/Parts|<font color="#ffffff">Parts</font>]]<br />
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== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.'''Chen GQ, Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''2.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from ''Alcaligenes eutrophus'' H16. Macromolecules. 19:2860-2864<br />
<br />
'''3.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53<br />
<br />
'''4.''' International Genetically Engineered Machines competition. 15 Jun 2008. 26 Jul 2008. <https://igem.org><br />
<br />
'''5.''' Holmes PA. 1985. Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''6.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208<br />
<br />
'''7.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports<br />
<br />
'''8.''' Lee SY, Choi J. 1999. Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151<br />
<br />
'''9.''' Lee SY. 1996. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
'''10.''' Poirier Y. 1999. Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
<br />
'''11.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page><br />
<br />
'''12.''' Shetty RP, Endy D, and TF Knight Jr. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5<br />
<br />
'''13.'''Schubert P, Kruger N, and Steinbuchel A. 1991. Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. The Journal of Bacteriology. 173(1): 168-175<br />
<br />
'''15.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:23:04Z<p>Liblint: /* References */</p>
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== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
7) Chen GQ, Wu Q. (2005). The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
20) Holmes PA. (1985). Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY, Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
31) Poirier Y. (1999). Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:22:36Z<p>Liblint: /* The Problem with PHB */</p>
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== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
7) Chen GQ, Wu Q. (2005). The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
20) Holmes PA. (1985). Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
31) Poirier Y. (1999). Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:21:43Z<p>Liblint: /* References */</p>
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY Choi J, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
7) Chen GQ, Wu Q. (2005). The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26:6565-6578<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
20) Holmes PA. (1985). Applications of PHB – a microbially produced biodegradable thermoplastic. Physics in technology. 16:32-36<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
31) Poirier Y. (1999). Green chemistry yields a better plastic. Nat. Biotechnol. 17:960-961<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:20:16Z<p>Liblint: /* PCR Primers */</p>
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== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY Choi J, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|800px|center]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:19:57Z<p>Liblint: /* PCR Primers */</p>
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<!--- The Mission, Experiments ---><br />
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY Choi J, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg|960px]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:19:46Z<p>Liblint: /* PCR Primers */</p>
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<!--- The Mission, Experiments ---><br />
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body {background-image:url("https://static.igem.org/mediawiki/2008/6/63/Bkgnd14.gif");background-repeat:repeat; }<br />
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<br />
<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY Choi J, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
''The figure below illustrates the greater binding affinity for primers without the prefix and suffix.''<br />
[[Image:USUprimers.jpg]]<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblinthttp://2008.igem.org/File:USUprimers.jpgFile:USUprimers.jpg2008-10-30T03:18:29Z<p>Liblint: </p>
<hr />
<div></div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:17:23Z<p>Liblint: /* Gel Electrophoresis */</p>
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY Choi J, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows a gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:16:58Z<p>Liblint: /* Gel Electrophoresis */</p>
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<!--- The Mission, Experiments ---><br />
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<br />
<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY Choi J, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
''Promoter PCR products were obtained through gel electrophoresis and a DNA purification kit. The image below shows one of the gels used to isolate these promoter regions.''<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblinthttp://2008.igem.org/File:USUpromotor.jpgFile:USUpromotor.jpg2008-10-30T03:14:44Z<p>Liblint: uploaded a new version of "Image:USUpromotor.jpg"</p>
<hr />
<div></div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:14:32Z<p>Liblint: /* Gel Electrophoresis */</p>
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY Choi J, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing.<br />
<br />
[[Image:USUpromotor.jpg|450px|center]]<br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblinthttp://2008.igem.org/File:USUpromotor.jpgFile:USUpromotor.jpg2008-10-30T03:13:12Z<p>Liblint: </p>
<hr />
<div></div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:11:32Z<p>Liblint: /* Results */</p>
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<!--- The Mission, Experiments ---><br />
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY Choi J, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing. <br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''''Detected intracellular PHB'''''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''''Successfully Created BioBricks'''''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''''BioBricks awaiting ligation'''''<br />
*PhaB in pSB1A3<br />
<br />
'''''BioBricks in earlier stages of completion:'''''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:10:58Z<p>Liblint: /* Results */</p>
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY Choi J, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing. <br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''Detected intracellular PHB'''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''Successfully Created BioBricks'''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''BioBricks awaiting ligation'''<br />
*PhaB in pSB1A3<br />
<br />
'''BioBricks in earlier stages of completion:'''<br />
*5’phaCpro2 in pSB1A3<br />
*-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:10:35Z<p>Liblint: /* Results */</p>
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<br />
<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY Choi J, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing. <br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''Detected intracellular PHB'''<br />
*1H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''Successfully Created BioBricks'''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''BioBricks awaiting ligation'''<br />
*PhaB in pSB1A3<br />
<br />
'''BioBricks in earlier stages of completion:'''<br />
*5’phaCpro2 in pSB1A3<br />
-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:10:16Z<p>Liblint: /* Results */</p>
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<br />
== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY Choi J, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing. <br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''Detected intracellular PHB'''<br />
*H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''Successfully Created BioBricks'''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''BioBricks awaiting ligation'''<br />
*PhaB in pSB1A3<br />
<br />
'''BioBricks in earlier stages of completion:'''<br />
*5’phaCpro2 in pSB1A3<br />
-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblinthttp://2008.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2008-10-30T03:09:58Z<p>Liblint: /* Conclusions */</p>
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== '''Abstract''' ==<br />
<br />
The Utah State University iGEM team project is focused on<br />
creating an efficient system for producing and monitoring PHA<br />
production in microorganisms. One goal of our research is to develop and<br />
optimize a method, using fluorescent proteins, for the detection of<br />
maximum product yield of polyhydroxybutyrate (PHB, a bioplastic) in<br />
recombinant E. coli and in ''Cupriavidus necator''. In order to develop an<br />
optimal PHB detection system, we worked to identify the<br />
most efficient reporter genes, and the best promoter sequences that<br />
would allow the GFP reporter to indicate maximum PHB production.<br />
<br />
== Project Objectives ==<br />
<br />
'''''DESIGN:''''' PHB reporter constructs (promoter region, reporter region, and PhaCAB cassette)<br />
<br />
'''''BUILD:''''' PHB reporter constructs<br />
<br />
'''''TEST:''''' functionality of constructs<br />
<br />
== Introduction ==<br />
<br />
=== Polyhydroxybutyrate ===<br />
[[Image:PHB1.jpg|250px|right]]<br />
Poly-β-hydroxybutyrate (PHB) is a low impact and naturally occurring biodegradable thermoplastic. It is intracellulary accumulated by a wide range of microorganisms as a carbon and energy reserve material in response to an environmental stress, such as nutrient limitation. PHB belongs to and is the most prevalent member of a broader class of polyesters called polyhydroxyalkanoates (PHA). Over 80 different PHAs have been classified, and each varies in mechanical properties. The difference within the polymers depends on the R side chain. Specifically, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biophotonic properties and biocompatibility with human tissues make it appealing for use in biomedical applications. Concerns related to limited oil supply, associated political issues, and public anxiety with landfills saturated with nonbiodegradable materials fuel the need for economical industrial PHB production (Lee, 1996; Poirier, 1999; Coats, 2005).<br />
<br />
=== The Problem with PHB===<br />
PHA research has increased in an effort to understand its potential, as well as to counteract some of the issues preventing its widespread use. Because of its high cost, PHB has been used mostly for specialized medical applications, including bone fixation, drug delivery systems and degradable sutures (Knowles, 1993: Kose, 2004; Holmes, 1985: Chen, 2005), and less for commercial packaging. <br />
<br />
High expense is the primary factor preventing industrial scale PHB production and commercialization. A low estimate for the cost of PHB production is $3-5/kg PHB versus $1/kg petroleum-based plastics (Choi, 1997). Some of the factors that contribute to this cost difference are reactor operation, substrate costs, and the extraction and downstream processing procedures (Coats 2005). There are many techniques that have been researched, or are currently being researched, to eliminate some of these major costs. Production methods for PHB involve the use of recombinant microorganisms, crops of transgenic PHB producing plants, and fermentation with naturally occurring strains of PHB accumulating organisms (Poirier, 1999). <br />
<br />
For this project, we attempted to minimize PHB production cost by considering the PHB yield, as this can drastically affect the polymer expense. In one example, a study showed that a PHB yield reduction from 88.3% CDW to 50% CDW contributed to a cost discrepancy of $1.6/kg (Lee SY Choi J, 1998). Recombinant ''E. coli'' cells harboring PHB production genes from ''C. necator'' were used in this project because it has been shown that these cells are capable of accumulating PHB in excess of 90% CDW. The goal of this study was to reduce the cost of industrial PHB production by creating a rapid biosensor system for determining the point when PHB accumulation is at its maximum, thereby optimizing polymer extraction.<br />
<br />
=== PHB Metabolic Pathways ===<br />
The metabolic pathway for PHB accumulation in ''C. necator'' involves three biosynthetic enzymes. The figure below shows the structure of the PHB operon. Transcription of these genes occurs under conditions of noncarbon nutrient limitation. <br />
[[Image:PHBCassette.jpg|900px]]<br><br />
<br />
<br />
<br />
[[Image:PHBpathway.jpg|300px|left]]<br />
<br />
'''Metabolic Pathway'''<br><br />
The metabolic pathway for PHB accumulation is depicted in the figure on the left and consists of three major steps (Verlinden, 2007).<br />
<br />
1. 3-ketothiolase (PhaA) produces acetoacetyl-CoA by joining two molecules of acetyl-CoA <br />
<br />
2. Acetoacetyl-CoA reductase (PhaB) promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA.<br />
<br />
3. 3-hydroxybutyryl-CoA is polymerized by PHB synthase (PhaC)<br />
<br />
Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria.<br><br />
<br />
<br />
<br />
<br />
=== Green Fluorescent Protein ===<br />
GFP (Green Fluorescent Protein) is a protein originally isolated from the jellyfish Aequorea victoria, fluorescing when exposed to ultraviolet light. GFP is used as a tag, attached to proteins as a marker. Only those cells in which the tagged gene is expressed will fluoresce. In such a way GFP acts as a positive marker of tranformation. In our laboratory, GFP is used as an expression reporter of PHB. The GFP gene primarily used in this project was GFP E0240 BioBrick supplied by iGEM.<br />
== Methods ==<br />
<br />
=== Organisms ===<br />
Two organisms were used as genetic sources for PHB: ''Cupriavidus necator'' and an ''Escherichia coli'' strain containing the PHB operon in a pBluescript vector. The E. coli strain proved to be easier to use as a PCR template because of the ability to miniprep the plasmid DNA to use as more pure template.<br />
<br />
===Transformations===<br />
A total of 26 transformations were successfully performed to become familiarized with the transformation process and to test different promoters and reporters. Three of these transformations were GPF plasmids with promoters of different strengths, taken from the iGEM promoter-testing kit. Another three of these transformations were BioBrick transformations. Top 10 competent E. coli cells were used for all transformations, excluding one transformation using a toxin-resistant strain. <br />
<br />
===Polymerase Chain Reaction===<br />
Nine sequences were targeted out of the PHB operon for amplification. Five PCR targets were promoter sequences: 5’phaCproF/ATG R, 5’phaCproF/SD R, 5’phaCproF/TC R, -35F/ATG R, and -35F/TC R. Four PCR targets were from phaCAB gene complex: phaC, phaA, phaB, and phaCAB. A variety of annealing temperatures and Mg++ concentrations were tried, and it was found that 60˚C annealing temperature and 12% (v/v) Mg++ concentration were optimal. Two sets of primers were used: those without the BioBrick prefix and suffix attached and those with them already attached. The use of the PCR conditions just described enabled the use of the latter, saving subsequent ligations from needing to be done. Such PCR reactions were successful with all of the promoter regions and the phaB gene.<br />
<br />
===PCR Primers===<br />
Two sets of primers were created for PCR – those containing the prefix and suffix restriction sites and those without the prefix and suffix. The primers with the prefix and suffix already attached were designed to save subsequent ligations of the prefix and suffix from needing to be done. The primers without the prefix and suffix had greater affinity to the DNA template than those with them because of the absence of non-binding segments.<br />
<br />
===Vector Selection===<br />
Two plasmids were primarily used during the course of the project – pSB1A3 and pSB3K3. pSB1A3 is an ampicillin-resistant plasmid, 2157 base pairs long, that was used as the vector for all submitted BioBricks. pSB3K3 is a Kanamycin-resistant plasmid, 2750 base pairs long, that was used for promoter testing in accordance with the iGEM promoter- testing protocol. <br />
<br />
=== Gel Electrophoresis ===<br />
DNA gel electrophoresis, 1% w/v agarose, was used for analysis of PCR products, digested plasmid, and ligation constructs. The cross-linked matrix formed by agarose gel separates DNA by size when an electrical field is created across the gel. DNA migrates toward the positive electrical anode by electromotive force because of the natural negative charge of the DNA’s phosphate-sugar backbone. Ethidium bromide and Sybr Green dyes selectively bind DNA and were used in various experiments for band observation. Select PCR, plasmid, and ligation products were excised from agarose gels for purification and further processing. <br />
<br />
=== GFP Correlation ===<br />
After some deliberation, it was decided to use Green Fluorescent Protein (GFP) as the PHB marker. Two methods were proposed to carry this through. The first method would involve ligating the GFP gene into the PHB operon, thereby allowing simultaneous transcription of the PHB-biosynthetic and GFP genes, regulated by the PHB-induction machinery. The second method would involve separate PHB-biosynthetic and GFP regions – either by bacteria containing two plasmids, one with the PHB operon and one with the GFP operon, or by using a plasmid containing both operons but in separate locations on the plasmid. In order to test for the most effective regions of the PHB promoter, five regions of the promoter were targeted for PCR (see PCR section). It was decided to then test these promoters using the iGEM promoter testing kit, testing for GFP expression in the pSB3K3 plasmid. A spectrofluorometer was used for fluorescence measurement.<br><br />
<br />
'''''PHB Promoter Testing'''''<br><br />
PHB promoter testing was planned according to the iGEM promoter testing kit instructions. Briefly, this kit contains three identical GFP-containing plasmids which only differ in their promoter strength (dubbed "weak," "medium," or "strong"). The kit outlined a method of preparing a promoterless GFP gene in a Kanamycin-resistant plasmid (pSB3K3). Our objective was to ligate our BioBrick promoter regions into this plasmid and compare them to the weak, medium, and strong promoter standards using a spectrofluorometer.<br />
<br />
===BioBrick Part Assembly===<br />
After performing PCR using suffix/prefix-containing primers, PCR products were digested with EcoR1 and Pst1. The samples were then run out on an electrophoresis gel, and the correctly-sized bands were cut out from the gel. The DNA was extracted using a Qiagen gel-purification kit. These inserts were then ligated into miniprepped pSB1A3 plasmid which had already been EcoR1/Pst1 digested and purified from a gel. These ligations were then transformed into Top 10 competent E. coli cells on Ampicillin-containing agar plates. If the transformations were successful, Amp-containing liquid cultures were made from which glycerol stocks and DNA minipreps were prepared. Portions of the minipreps were digested with restriction enzymes from the prefix and the suffix, and the samples were run out on a gel to test for expected insert and vector lengths. If this test was passed, the sample was submitted as a BioBrick.<br />
<br />
== Results ==<br />
<br />
'''Detecting intracellular PHB'''<br />
*H-NMR was successfully used to detect PHB accumulation in recombinant ''E. coli'' harboring the PHB-biosynthetic genes from ''C. necator''.<br />
<br />
'''Successfully Created BioBricks'''<br />
*5’phaCpro1 in pSB1A3<br />
*5’phaCpro3 in pSB1A3<br />
*-35/TC in psB1A3<br />
<br />
'''BioBricks awaiting ligation'''<br />
*PhaB in pSB1A3<br />
<br />
'''BioBricks in earlier stages of completion:'''<br />
*5’phaCpro2 in pSB1A3<br />
-35Pro in pSB1A3<br />
*PhaA in pSB1A3<br />
*PhaC in pSB1A3<br />
*PhaCAB in pSB1A3<br />
<br />
== Conclusions ==<br />
'''''PHB accumulation needs to be monitored.'''''<br> Polyhydroxybutyrate has great potential as a renewable, biodegradable plastic. At present, extraction methods of PHB are more costly than production methods for petrochemically-derived plastics, making higher extraction efficiency a necessity. Using a genetic marker to identify optimal extraction time will reduce these costs by providing maximum PHB yields. <br />
<br />
'''''GFP could be an effective marker.'''''<br> Green fluorescent protein has been shown to be an effective genetic marker since its discovery in 1968. Since it's genetic sequence and optical properties have been well studied, the USU iGEM team felt GFP would provide acceptable indication of PHB expression.<br />
<br />
'''''The USU iGEM team has constructed some necessary BioBricks.'''''<br> The effective use of GFP as an expression marker will depend on sufficient understanding of both the PHB promoter and PhaCAB cassette. The USU iGEM team has made progress in both of these areas and has created three BioBricks from the promoter region.<br />
<br />
== References ==<br />
'''1.''' Doi Y, Kunioka M, Nakamura Y, Soga K. 1986. Nuclear magnetic resonance studies on poly(B-hydroxybutyrate) and a copolyester of B-hydroxybutyrate and B-hydroxyvalerate isolated from Alcaligenes eutrophus H16. Macromolecules. 19:2860-2864.<br />
<br />
'''2.''' Endy D. 2005. Foundations for engineering biology. Nature. 438(7067):449-53.<br />
International Genetically Engineered Machines competition. 17 Oct 2008. 26 Jul 2008. <https://igem.org>.<br />
<br />
'''3.''' Kang Z, Wang Q, and H Zhang. 2008. Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production. Biotechnological Products and Process Engineering. 79:203-208.<br />
<br />
'''4.''' Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. Tech. rep., MIT Synthetic Biology Working Group Technical Reports.<br />
<br />
25) Lee SY Choi J. (1999). Metabolic engineering strategies for the production of polyhydroxyalkanoates, a family of biodegradable polymers. eds. S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, USA, pp. 113-151.<br />
26) Lee SY. (1996). Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering. 49:1-14<br />
<br />
<br />
'''5.''' Registry of Standard Biological Parts. 17 Oct 2008. 26 Jul 2008 <http://partsregistry.org/Main_Page>.<br />
<br />
'''6.''' Shetty RP, Endy D, Knight Jr TF. 2008. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2:5.<br />
<br />
'''7.''' Verlindin RAJ, Hill DJ, Kenward MA, Williams CD, and I Radecka. 2007. Bacterial synthesis of biodegradable Polyhydroxyalkanoates. Journal of Applied Microbiology 102:1437–1449.</div>Liblint