http://2008.igem.org/wiki/index.php?title=Special:Contributions/Nathan.puhl&feed=atom&limit=50&target=Nathan.puhl&year=&month=2008.igem.org - User contributions [en]2024-03-28T09:08:15ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Team:University_of_Lethbridge/ProjectTeam:University of Lethbridge/Project2008-10-30T01:33:25Z<p>Nathan.puhl: </p>
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<div>__NOTOC__<br />
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<span style="background-color:#000000; color:white"><!--__TOC__--></span><br />
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==<span style="background-color:#000000; color:white"> '''The “Bacuum" Cleaner – an intelligent self-propelling keener cleaner'''</span> ==<br />
<br />
<!--- The Mission, Experiments ---><br />
Our goal is to create a modified ''Escherichia coli'' bacterium capable of seeking out and degrading toxic aromatic pollutants created during the oil refinery and mining processes. Our “Bacuum" cleaner will respond to this destructive compound through interaction with a programmed riboswitch (an mRNA capable of binding a target molecule with its 5’ UTR which causes a conformational change, thereby regulating any downstream protein translation). By using riboswitches that switch at varying concentrations of target ligand, we can alter the signal which is induced. At low concentrations, we intend to have our riboswitch express the motility protein cheZ in ''E. coli'', thus directing the bacterium towards higher concentrations of our target molecule (i.e. positive chemotaxis). Once it reaches a threshold concentration, a catabolic pathway capable of degrading our target pollutant will be activated.<br />
<br />
We plan to use [http://en.wikipedia.org/wiki/Systematic_Evolution_of_Ligands_by_Exponential_Enrichment <font color=yellow>SELEX</font>] to reprogram the theophylline riboswitch initially characterized by our team last year. For our proof-of-principle concept, we chose 2-chlorobenzoate, a compound related to polychlorinated biphenyls (PCBs), as our target molecule. This not only builds on our past work with riboswitches but also demonstrates an alternative function for riboswitch-controlled chemotaxis, changing from a simple detection to active “search and destroy” role. Fluorescent protein expression will be used to demonstrate and characterize the functionality of the riboswitch.<br />
<br />
Our project also has major environmental implications, especially in Alberta, where the oil industry is the driving economic sector. The tailings ponds used to house discarded mining refuse from the oil refineries pose a major environmental dilemma. The problem arises with being unable to collect and degrade the compounds which make up these toxic soups. These not only toxic but often corrosive water beds not only affect the local environment but can also cause severe repercussions to other ecosystems as much of the wildlife relies on getting their food and water from the now disrupted bionetwork. These tailings ponds are often laced with heavy metals and dangerous chemicals, including these toxic aromatic hydrocarbons which can be extremely difficult to breakdown. Despite the efforts of the corporations responsible, some of these chemicals can leech into and contaminate the surrounding ground water and soil. It is our intention that our “Bacuum” will not only remove these compounds from such an aqueous system, but will also eliminate them. This goes one step further than the ordinary less-intelligent bag-dependent vacuum cleaners. Ultimately we aim to have our bacteria convert the target aromatic into a useable compound. One example would be the biosynthesis of fatty acids as an alternative fuel source, creating a potential “Bioreactor” which is capable of creating a more environmentally friendly fuel source.<br />
<br />
== <span style="background-color:#000000; color:white">Project Details</span>==<br />
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=== <span style="background-color:#000000; color:white">Chemotaxis - the "search" from "search and destroy" </span>===<br />
<br />
Our subproject is to ensure our 'bacuum cleaner' will search out and move towards our potential target. Using a riboswitch which responds to this target, a gene involved in motility in ''Escherichia coli'' (''CheZ'') will be switched to an 'on' state (Topp and Gallivan, 2007). ''CheZ'' controls bacterial movement by dephosphorylating ''CheY'', altering the bacteria from random tumbling to directed movement. By doing so, we hope to observe the bacteria moving towards the target molecule in a positive chemotaxis manner. <br />
<br />
Because we wish to eventually have our bacteria degrade this activating compound, we need to have a multi-level response system established. Our bacteria should respond to low levels of the target by activating our ''CheZ'' riboswitch (through a strong binding aptamer) and to high levels by activating the catabolic pathway responsible for its degradation. By doing so, we will have created our 'search and destroy' system.<br />
<br />
Our subgroup will attempt to establish a working motility assay to prove control of chemotaxis is possible with an already characterized theophylline-binding riboswitch and later with our new target molecule. We will also use a colour read-out system of two fluorescent proteins to demonstrate the differential binding of the ligand at varying concentrations.<br />
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=== <span style="background-color:#000000; color:white">Riboswitch Characterization</span>===<br />
<br />
This subproject was created to quantify and characterize the binding affinity of the theophylline riboswitch to theophylline as well as other similar aromatic ligands. We will use Green Fluorescent Protein (GFP) sequenced behind the riboswitch to help in this endeavour. When the target ligand is bound to the riboswitch, the production of GFP will be induced, producing green colonies. When the ligand is not bound, GFP will not be produced. We intend to try to characterize the riboswitch with and without the iGEM scar to determine if this produces any effect on the relative effeciency of the riboswitch on the protein.<br />
<br />
We will use variable concentrations of Theophylline, Caffeine and 3-methylxanthine to help with the characterization of the initial theophylline riboswitch. We will also test other aromatic compounds to determine wether or not, the theophylline riboswitch can be utilized to bind them as well. <br />
<br />
Once we have successfully produced mutant riboswitches through SELEX, will we then perform these same characterization experiments to determine their binding affinities.<br />
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[[Image:riboswitch.jpeg|650px|center]]<br />
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===<span style="background-color:#000000; color:white"> SELEX </span>===<br />
<br />
Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a method used to produce novel nucleic acid sequences, referred to as aptamers, that bind specific ligands. SELEX involves screening a library of possible aptamers for binding capability repeatedly until a sequence of the desired specificity or binding affinity is found.<br />
<br />
SELEX will be used in our project to produce multiple riboswitches capable of specifically binding the target toxin at varying concentrations. Multiple riboswitches are needed to allow a separation between the "search" and the "destroy" functions of our Bacuum cleaner, with the CheZ search ability controlled at low ligand levels and the degradation pathway controlled at high levels.<br />
<br />
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=== <span style="background-color:#000000; color:white">TIR </span>===<br />
<br />
Translation initiation regions (TIRs) are elements found in certain eukaryotic mRNAs that facilitate translation initiation by noncanonical, end independent interactions (Hellen, 2007; Skorski et al., 2006). We are going to incorporate the TIR from the ''rpsA'' gene, encoding ribosomal protein S1, of ''E. coli'' into our "Bacuum cleaner" with green fluorescent protein as a means of monitoring the location of our cells using minimal cell resources. <br />
<br />
The TIR from the rpsA gene will be characterized through the comparison of translation rates for GFP to other canonical RBS. This will allow us to standardize the number of ribosomal initiations per second (RiPS). In generating these parameters we will be able to contribute to building a database of standard parts that can be used to generate biological systems that have a greater degree of reliability.<br />
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===<span style="background-color:#000000; color:white"> 2-chlorobenzoate metabolism </span>===<br />
<br />
Polychlorinated biphenyls (PCBs) have been established as toxic and environmentally unfriendly since the early 1980s. One potential intermediate step in the degradation of PCBs involves elimination of the chloro substituents. A dehalogenation pathway for ortho-halobenzoates was discovered in ''Pseudomonas aeruginosa'' strain 142 and successfully isolated and characterized by Tsoi et al. (1999). This metabolic pathway, comprising of the ''ohb'' genes, catalyzes the degradation of 2-chlorobenzoate into catechol. We aim to combine this system with the catechol 2,3 dioxygenase ''xylE'' gene (Ingram et al., 1989) to further break down catechol into the highly-coloured 2-hydroxymuconic semialdehyde. By doing so, we will combine our degradation system with a reporter system, bringing yet another level of intelligence to our incredibly awesome “bacuum cleaner.”<br />
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== <span style="background-color:#000000; color:white">References</span>==<br />
<br />
Hellen, C. U. T. 2007. Bypassing translation initiation. ''Structure'' 15, 4-6.<br />
<br />
Ingram, C., Brawner, M., Youngman, P., and Westpheling, J. 1989. ''xylE'' functions as an efficient reporter gene in ''Streptomyces'' spp.: Use for the study of ''galPI'', a catabolite-controlled promoter. ''J. Bacteriol.'' 171, 6617-6624.<br />
<br />
Jenison, R. D., Gill, S. C., Pardi, A., and Polinsky, B. 1994. High resolution molecular discrimination by RNA. ''Science'' 263, 1425-1429.<br />
<br />
Lynch, S. A., Desai, S. K., Sajja, H. K., and Gallivan J. P. A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function. ''Chem. Biol.'' 14, 173-184.<br />
<br />
Nomura, Y. and Yokobayashi, Y. Dual selection of a genetic switch by a single selection marker. ''BioSystems'' 90, 115-120. <br />
<br />
Skorski, P., Leroy, P., Fayet, O., Dreyfus, M., and Denmat, S. H. 2006. The highly efficient translation initiation region from the Escherichia coli rpsA gene lacks a Shine Delgarno element. ''J. Bacteriol.'' 188, 6277-6285.<br />
<br />
Topp, S. and Gallivan, J. 2007. Guiding bacteria with small molecules and RNA. ''J. Am. Chem. Soc.'' 129, 6807-6811.<br />
<br />
Tsoi, T. V., Plotnikova, E. G., Cole, J. R., Guerin, W. F., Bagdasarian, M. and Tiedje, J. M. 1999. Cloning, expression, and nucleotide sequence of the ''Pseudomonas aeruginosa'' 142 ''ohb'' genes coding for oxygenolytic ''ortho'' dehalogenation of halobenzoates. ''Appl. Environ. Microbiol.'' 65, 2151-2161.<br />
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</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/PartsTeam:University of Lethbridge/Parts2008-10-30T01:22:03Z<p>Nathan.puhl: /* Parts Added to the Registry */</p>
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=<span style="background-color:#000000; color:white">Parts Added to the Registry</span>=<br />
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CheZ - BBa_K147000<br />
<br />
CheZ is an enzyme invovled in E.coli chemotaxis, dephosphorylating CheY which causes the flagellum to reverse rotation and produce forward thrust. By regulating CheZ expression in the CheZ knockout strain RP1616, cell motility can be directed up a chemical gradient of choice.<br />
<br />
xylE - BBa_K147003<br />
<br />
The xylE gene from Pseudomonas putida encodes the enzyme catechol 2,3-dioxygenase which catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. This reaction results in a vibrant yellow color in the cell.<br />
<br />
ohb A - BBa_K147002<br />
<br />
The ohb A gene produces a subunit of the ortho-halobenzoate 1,2-dioxygenase found in Pseudomonas aeruginosa. This complex catalyzes the conversion of 2-chlorobenzoate into catechol <br />
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</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/PartsTeam:University of Lethbridge/Parts2008-10-30T01:20:15Z<p>Nathan.puhl: /* Parts Added to the Registry */</p>
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=<span style="background-color:#000000; color:white">Parts Added to the Registry</span>=<br />
<br />
CheZ - BBa_K147000<br />
<br />
CheZ is an enzyme invovled in E.coli chemotaxis, dephosphorylating CheY which causes the flagellum to reverse rotation and produce forward thrust. By regulating CheZ expression in the CheZ knockout strain RP1616, cell motility can be directed up a chemical gradient of choice.<br />
<br />
xylE - BBa_K147003<br />
<br />
The xylE gene from Pseudomonas putida encodes the enzyme catechol 2,3-dioxygenase which catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. This reaction results in a vibrant yellow color in the cell.<br />
<br />
ohb A - BBa_K147002<br />
<br />
The ohb A gene produces a subunit of the ortho-halobenzoate 1,2-dioxygenase found in Pseudomonas aeruginosa. This complex catalyzes the conversion of 2-clorobenzoate into catechol <br />
<br />
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</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/PartsTeam:University of Lethbridge/Parts2008-10-30T01:18:19Z<p>Nathan.puhl: /* Parts Added to the Registry */</p>
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<div>__NOTOC__<br />
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=<span style="background-color:#000000; color:white">Parts Added to the Registry</span>=<br />
<br />
CheZ - BBa_K147000<br />
<br />
CheZ is an enzyme invovled in E.coli chemotaxis, dephosphorylating CheY which causes the flagellum to reverse rotation and produce forward thrust. By regulating CheZ expression in the CheZ knockout strain RP1616, cell motility can be directed up a chemical gradient of choice.<br />
<br />
ohb A - BBa_K147002<br />
<br />
The ohb A gene produces a subunit of the ortho-halobenzoate 1,2-dioxygenase found in Pseudomonas aeruginosa. This complex catalyzes the conversion of 2-clorobenzoate into catechol <br />
<br />
xylE - BBa_K147003<br />
<br />
The xylE gene from Pseudomonas putida encodes the enzyme catechol 2,3-dioxygenase which catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. This reaction results in a vibrant yellow color in the cell.<br />
<br />
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</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/PartsTeam:University of Lethbridge/Parts2008-10-30T01:17:14Z<p>Nathan.puhl: /* Parts Added to the Registry */</p>
<hr />
<div>__NOTOC__<br />
<br />
<div style="background-color:#000000; color:white"><br />
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=<span style="background-color:#000000; color:white">Parts Added to the Registry</span>=<br />
<br />
CheZ - <br />
<br />
CheZ is an enzyme invovled in E.coli chemotaxis, dephosphorylating CheY which causes the flagellum to reverse rotation and produce forward thrust. By regulating CheZ expression in the CheZ knockout strain RP1616, cell motility can be directed up a chemical gradient of choice.<br />
<br />
ohb A - <br />
<br />
The ohb A gene produces a subunit of the ortho-halobenzoate 1,2-dioxygenase found in Pseudomonas aeruginosa. This complex catalyzes the conversion of 2-clorobenzoate into catechol <br />
<br />
xylE - <br />
<br />
The xylE gene from Pseudomonas putida encodes the enzyme catechol 2,3-dioxygenase which catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. This reaction results in a vibrant yellow color in the cell.<br />
<br />
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</div></div>Nathan.puhlhttp://2008.igem.org/Jamboree/Schedule/Practice_sessionsJamboree/Schedule/Practice sessions2008-10-08T01:55:02Z<p>Nathan.puhl: /* Friday November 7 : Practice Talks sign-up sheet */</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 />
<br />
<html><br />
<link rel="stylesheet" href="http://parts.mit.edu/igem07/index.php?title=User:Macowell/schedule.css&action=raw&ctype=text/css"><br />
<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>B1</td><br />
<td>C1</td><br />
<td>D1</td><br />
<td>E1</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>C2</td><br />
<td>D2</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>C3</td><br />
<td>D3</td><br />
<td>NYMU-Taipei</td><br />
<td>F3</td><br />
<td>G3</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>C4</td><br />
<td>D4</td><br />
<td>E4</td><br />
<td>F4</td><br />
<td>G4</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>C5</td><br />
<td>D5</td><br />
<td>E5</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>A6</td><br />
<td>Alberta_NINT</td><br />
<td>ULeth</td><br />
<td>D6</td><br />
<td>E6</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>A7</td><br />
<td>Waterloo</td><br />
<td>C7</td><br />
<td>D7</td><br />
<td>E7</td><br />
<td>F7</td><br />
<td>G7</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>A8</td><br />
<td>B8</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>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/ProjectTeam:University of Lethbridge/Project2008-08-24T23:48:59Z<p>Nathan.puhl: /* References */</p>
<hr />
<div>__NOTOC__<br />
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<span style="background-color:#000000; color:white"><!--__TOC__--></span><br />
<br />
==<span style="background-color:#000000; color:white"> '''The “Bacuum" Cleaner – an intelligent self-propelling keener cleaner'''</span> ==<br />
<br />
<!--- The Mission, Experiments ---><br />
Our goal is to create a modified ''Escherichia coli'' bacterium capable of seeking out and degrading toxic aromatic pollutants created during the oil refinery and mining processes. Our “Bacuum" cleaner will respond to this destructive compound through interaction with a programmed riboswitch (an mRNA capable of binding a target molecule with its 5’ UTR which causes a conformational change, thereby regulating any downstream protein translation). By using riboswitches that switch at varying concentrations of target ligand, we can alter the signal which is induced. At low concentrations, we intend to have our riboswitch express the motility protein cheZ in ''E. coli'', thus directing the bacterium towards higher concentrations of our target molecule (i.e. positive chemotaxis). Once it reaches a threshold concentration, a catabolic pathway capable of degrading our target pollutant will be activated.<br />
<br />
We plan to use [http://en.wikipedia.org/wiki/Systematic_Evolution_of_Ligands_by_Exponential_Enrichment <font color=yellow>SELEX</font>] to reprogram the theophylline riboswitch initially characterized by our team last year. For our proof-of-principle concept, we chose 2-chlorobenzoate, a compound related to polychlorinated biphenyls (PCBs), as our target molecule. This not only builds on our past work with riboswitches but also demonstrates an alternative function for riboswitch-controlled chemotaxis, changing from a simple detection to active “search and destroy” role. Fluorescent protein expression will be used to demonstrate and characterize the functionality of the riboswitch.<br />
<br />
Our project also has major environmental implications, especially in Alberta, where the oil industry is the driving economic sector. The tailings ponds used to house discarded mining refuse from the oil refineries pose a major environmental dilemma. The problem arises with being unable to collect and degrade the compounds which make up these toxic soups. These not only toxic but often corrosive water beds not only affect the local environment but can also cause severe repercussions to other ecosystems as much of the wildlife relies on getting their food and water from the now disrupted bionetwork. These tailings ponds are often laced with heavy metals and dangerous chemicals, including these toxic aromatic hydrocarbons which can be extremely difficult to breakdown. Despite the efforts of the corporations responsible, some of these chemicals can leech into and contaminate the surrounding ground water and soil. It is our intention that our “Bacuum” will not only remove these compounds from such an aqueous system, but will also eliminate them. This goes one step further than the ordinary less-intelligent bag-dependent vacuum cleaners. Ultimately we aim to have our bacteria convert the target aromatic into a useable compound. One example would be the biosynthesis of fatty acids as an alternative fuel source, creating a potential “Bioreactor” which is capable of creating a more environmentally friendly fuel source.<br />
<br />
== <span style="background-color:#000000; color:white">Project Details</span>==<br />
<br />
<br />
<br />
<br />
<br />
=== <span style="background-color:#000000; color:white">Chemotaxis - the "search" from "search and destroy" </span>===<br />
<br />
Our subproject is to ensure our 'bacuum cleaner' will search out and move towards our potential target. Using a riboswitch which responds to this target, a gene involved in motility in ''Escherichia coli'' (''CheZ'') will be switched to an 'on' state (Topp and Gallivan, 2007). ''CheZ'' controls bacterial movement by dephosphorylating ''CheY'', altering the bacteria from random tumbling to directed movement. By doing so, we hope to observe the bacteria moving towards the target molecule in a positive chemotaxis manner. <br />
<br />
Because we wish to eventually have our bacteria degrade this activating compound, we need to have a multi-level response system established. Our bacteria should respond to low levels of the target by activating our ''CheZ'' riboswitch (through a strong binding aptamer) and to high levels by activating the catabolic pathway responsible for its degradation. By doing so, we will have created our 'search and destroy' system.<br />
<br />
Our subgroup will attempt to establish a working motility assay to prove control of chemotaxis is possible with an already characterized theophylline-binding riboswitch and later with our new target molecule. We will also use a colour read-out system of two fluorescent proteins to demonstrate the differential binding of the ligand at varying concentrations.<br />
<br />
=== <span style="background-color:#000000; color:white">Riboswitch Characterization</span>===<br />
<br />
This subproject was created to quantify and characterize the binding affinity of the theophylline riboswitch to theophylline as well as other similar aromatic ligands. We will use Green Fluorescent Protein (GFP) sequenced behind the riboswitch to help in this endeavour. When the target ligand is bound to the riboswitch, the production of GFP will be induced, producing green colonies. When the ligand is not bound, GFP will not be produced. We intend to try to characterize the riboswitch with and without the iGEM scar to determine if this produces any effect on the relative effeciency of the riboswitch on the protein.<br />
<br />
We will use variable concentrations of Theophylline, Caffeine and 3-methylxanthine to help with the characterization of the initial theophylline riboswitch. We will also test other aromatic compounds to determine wether or not, the theophylline riboswitch can be utilized to bind them as well. <br />
<br />
Once we have successfully produced mutant riboswitches through SELEX, will we then perform these same characterization experiments to determine their binding affinities.<br />
<br />
[[Image:riboswitch.jpeg|650px|center]]<br />
<br />
===<span style="background-color:#000000; color:white"> SELEX </span>===<br />
<br />
Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a method used to produce novel nucleic acid sequences, referred to as aptamers, that bind specific ligands. SELEX involves screening a library of possible aptamers for binding capability repeatedly until a sequence of the desired specificity or binding affinity is found.<br />
<br />
SELEX will be used in our project to produce multiple riboswitches capable of specifically binding the target toxin at varying concentrations. Multiple riboswitches are needed to allow a separation between the "search" and the "destroy" functions of our Bacuum cleaner, with the CheZ search ability controlled at low ligand levels and the degradation pathway controlled at high levels.<br />
<br />
=== <span style="background-color:#000000; color:white">TIR </span>===<br />
<br />
Translation initiation regions (TIRs) are elements found in certain eukaryotic mRNAs that facilitate translation initiation by noncanonical, end independent interactions (Hellen, 2007; Skorski et al., 2006). We are going to incorporate the TIR from the ''rpsA'' gene, encoding ribosomal protein S1, of ''E. coli'' into our "Bacuum cleaner" with green fluorescent protein as a means of monitoring the location of our cells using minimal cell resources. <br />
<br />
The TIR from the rpsA gene will be characterized through the comparison of translation rates for GFP to other canonical RBS. This will allow us to standardize the number of ribosomal initiations per second (RiPS). In generating these parameters we will be able to contribute to building a database of standard parts that can be used to generate biological systems that have a greater degree of reliability.<br />
<br />
===<span style="background-color:#000000; color:white"> Metabolism of 2-chlorobenzoate </span>===<br />
<br />
Polychlorinated biphenyls (PCBs) have been established as toxic and environmentally unfriendly since the early 1980s. One potential intermediate step in the degradation of PCBs involves elimination of the chloro substituents. A dehalogenation pathway for ortho-halobenzoates was discovered in ''Pseudomonas aeruginosa'' strain 142 and successfully isolated and characterized by Tsoi et al. (1999). This metabolic pathway, comprising of the ''ohb'' genes, catalyzes the degradation of 2-chlorobenzoate into catechol. We aim to combine this system with the catechol 2,3 dioxygenase ''xylE'' gene (Ingram et al., 1989) to further break down catechol into the highly-coloured 2-hydroxymuconic semialdehyde. By doing so, we will combine our degradation system with a reporter system, bringing yet another level of intelligence to our incredibly awesome “bacuum cleaner.”<br />
<br />
== <span style="background-color:#000000; color:white">Results </span>==<br />
<br />
<br />
== <span style="background-color:#000000; color:white">References</span>==<br />
<br />
Hellen, C. U. T. 2007. Bypassing translation initiation. ''Structure'' 15, 4-6.<br />
<br />
Ingram, C., Brawner, M., Youngman, P., and Westpheling, J. 1989. ''xylE'' functions as an efficient reporter gene in ''Streptomyces'' spp.: Use for the study of ''galPI'', a catabolite-controlled promoter. ''J. Bacteriol.'' 171, 6617-6624.<br />
<br />
Jenison, R. D., Gill, S. C., Pardi, A., and Polinsky, B. 1994. High resolution molecular discrimination by RNA. ''Science'' 263, 1425-1429.<br />
<br />
Lynch, S. A., Desai, S. K., Sajja, H. K., and Gallivan J. P. A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function. ''Chem. Biol.'' 14, 173-184.<br />
<br />
Nomura, Y. and Yokobayashi, Y. Dual selection of a genetic switch by a single selection marker. ''BioSystems'' 90, 115-120. <br />
<br />
Skorski, P., Leroy, P., Fayet, O., Dreyfus, M., and Denmat, S. H. 2006. The highly efficient translation initiation region from the Escherichia coli rpsA gene lacks a Shine Delgarno element. ''J. Bacteriol.'' 188, 6277-6285.<br />
<br />
Topp, S. and Gallivan, J. 2007. Guiding bacteria with small molecules and RNA. ''J. Am. Chem. Soc.'' 129, 6807-6811.<br />
<br />
Tsoi, T. V., Plotnikova, E. G., Cole, J. R., Guerin, W. F., Bagdasarian, M. and Tiedje, J. M. 1999. Cloning, expression, and nucleotide sequence of the ''Pseudomonas aeruginosa'' 142 ''ohb'' genes coding for oxygenolytic ''ortho'' dehalogenation of halobenzoates. ''Appl. Environ. Microbiol.'' 65, 2151-2161.<br />
<br />
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</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabAugustTeam:University of Lethbridge/Notebook/GeneralLabAugust2008-08-24T23:42:55Z<p>Nathan.puhl: /* Christa, Munima, Nathan Puhl, Roxanne */</p>
<hr />
<div>(Aug. 1- 7 belong in other August notebooks)<br />
===August 1, 2008===<br />
====Nathan Puhl, Roxanne====<br />
Riboswitch 20 uL PCR. Set up 4 reactions (25 uL for each total volume). 1 uL or 1/100 pTopp and 1 uL or H1/100 PCR from July 28, 2008. <br />
<br />
PCR conditions:<br />
A. Initial denaturation: 98 C (3 min)<br />
B. -Denaturation: 98 C (10 sec)<br />
- Annealing: 55 C (30 sec)<br />
-Extension: 72 C (15 sec)<br />
-30 cycles<br />
C. Final extension: 72 C (7 min)<br />
<br />
===August 2, 2008===<br />
====Nathan Puhl, Roxanne====<br />
Ran riboswitch (Aug. 1, 2008) on 3% agarose gel. Results are in the hard copy lab notebook. Looks like 76 bp band will extract.<br />
<br />
===August 5, 2008===<br />
====Nathan Puhl et al====<br />
Gel extracted two bands really closer to each other from PCR (August 1) at ~76 bp. Used Qiagen MiniElute kit. Results are in the hard copy lab notebook. Seems as if gel extraction was successful.<br />
____<br />
2 color reporter system:<br />
<br />
Set up digest of LacI and DT.<br />
<br />
LacI - EcoRI and SpeI<br />
<br />
DT - EcoRI and XbaI<br />
<br />
Reaction mixture:<br />
-5 uL template<br />
-5 uL NEB 2 Buffer<br />
-0.5 uL BSA?<br />
-1 uL RE #1<br />
-1 uL RE #2<br />
- 37.5 uL ddH20<br />
<br />
Left at 37 C overnight. Heat deactivation (65 C) for 15 minutes.<br />
<br />
<br />
===August 6, 2008===<br />
====Nathan Puhl, Roxanne====<br />
2 color reporter system:<br />
<br />
Gel extracted LacI insert and DT vector. Didn't run gel yet.<br />
<br />
===August 7, 2008===<br />
<br />
<br />
<br />
====Nathan Puhl, Roxanne====<br />
Riboswitch:<br />
<br />
Set up PCR using purified riboswitch from Aug. 5, 2008 with platinum Taq (50 uL reaction). Made Master Mix for three reactions.<br />
Master Mix:<br />
-10x Buffer (no Mg2+): 15 uL<br />
-10 mM dNTPs: 3 uL<br />
-50 mM Mg2+: 4.5 uL<br />
-10uM RF: 3 uL<br />
-10uM RR: 3 uL<br />
-Plat. poly: 0.6 iL<br />
-H20: 120.9 uL<br />
-template: 1 uL<br />
<br />
Cycle conditions:<br />
A. Initial denaturation: 94 C (2 min)<br />
B. -Denaturation: 94 C (30 sec)<br />
- Annealing: 55 C (30 sec)<br />
-Extension: 72 C (30 sec)<br />
-30 cycles<br />
C. Final extension: 72 C (7 min)<br />
<br />
====Roxanne====<br />
-Ran the PCR Product on a 3% gel using only 1uL of DNA from the riboswitch.<br />
<br />
===August 13, 2008===<br />
====Nathan Puhl and Sebastian====<br />
Verified and quantified the LacI and DT restriction digest products.<br />
<br />
Set up a Ligase experiment for LacI and DT<br />
- 1.3 uL vector(DT)<br />
- 8 uL insert(LacI)<br />
- 2 uL 10x Ligase buffer<br />
- 1.5 uL Ligase<br />
- 7.2 uL ddH2O<br />
- 20 uL total volume<br />
<br />
<br />
===August 14, 15===<br />
====Nathan Puhl, Munima, Selina====<br />
Poured 61 LB + Amp plates. Stored in iGEM 4 C fridge.<br />
<br />
<br />
===August 15, 2008===<br />
====Roxanne====<br />
<br />
Used Qiagen Plasmid MiniPrep Kit to Plasmid Prep Last Year's Biobrick Parts that were Incubated overnight.<br />
<br />
Ran Plasmids in a 1% Agarose Gel at 100 V for 30 minutes.<br />
<br />
===August 16, 2008===<br />
<br />
====Nathan Puhl, Roxanne, Munima====<br />
-Restriction Digested the purified riboswitch and pSB1A7 with XbaI and SpeI, let it run for 4 hours<br />
<br />
<br />
====Nathan Puhl, Roxanne====<br />
-Ran all of the restricted pSB1A7 plasmid through a 1% agarose gel at 100V for 25 minutes.<br />
-Ran a gel extraction on the pSB1A7 cut plasmid, and ran a PCR clean-up reaction on the digested RS1 and RS2 amplicons.<br />
-Ran 1 uL of each on a 1% to quantify the amount of DNA present.<br />
-Ligated RS1 + pSB1A7, and RS2 + pSB1A7, using T4 DNA Ligase.<br />
<br />
-1 uL of RS1/RS2<br />
-4 uL of pSB1A7<br />
-1 uL of 10x T4 DNA Ligase Buffer<br />
-0.33 uL of T4 DNA Ligase<br />
-3.67 uL of water<br />
<br />
allowed the reaction to go overnight<br />
<br />
<br />
===August 17, 2008===<br />
====Nathan Puhl====<br />
-Transformed DH5a cells with the pSB1A7 + RS1, and pSB1A7 + RS2 plasmids.<br />
<br />
-Plated on semi-solid agar plates containing 100 ug/mL of ampicillin.<br />
<br />
===August 18===<br />
====Christa, Nathan Puhl, Munima====<br />
-Nathan checked the plates for growth. Colonies are present.<br />
<br />
-Ran a colony PCR of the pSB1A7 + RS1, and pSB1A7 + RS2 recombinant cells transformed by Nathan and Roxanne on August 17.<br />
<br />
-Inoculated the cells into tubes of liquid media + 100 ug/ml of ampicillin.<br />
<br />
Roxanne will remove the PCR tube from the thermocycler in the morning.<br />
<br />
===August 19, 2008===<br />
<br />
====Roxanne====<br />
-Ran the PCR Product on a 2% Agarose Gel at 100 V for 33 minutes.<br />
<br />
[[Image:RS1 RS2 gel.jpg| 350 px]]<br />
<br />
-Plasmid Prepped and made glycerol stocks from the RS1-1 and RS2-1 tubes of cells incubated in LB media + 100 ug/mL ampicillin.<br />
<br />
Ran the pRS1 and pRS2 plasmids on a 1% Agarose Gel at 100 V for 30 minutes.<br />
<br />
[[Image:pRS1 pRS2 gel.jpg| 150 px]]<br />
<br />
====Christa, Munima, Sebastian====<br />
Did a plasmid prep on pSB1A7 using QIAprep Spin Miniprep Kit. Stocked 4- 50uL of pSB1A7 and stored in iGEM -20 C.<br />
<br />
====Sebastian, Nathan Puhl====<br />
Ran products from plasmid prep (pSB1A7 x 4 samples) on 1% agarose gel for 27 minutes.<br />
<br />
-Lane 1 - 1 kb GeneRuler ladder (2 uL)<br />
-Lane 2 -6 pSB1A7 (3 uL) + 6x loading dye (2 uL); Mixed up what sample was in Lane 4, so Lanes 5 and 6 were run.<br />
<br />
Conclusion: The bands appeared to be at the correct size for pSB1A7.<br />
<br />
===August 21===<br />
====Nathan Puhl, Roxanne====<br />
-Screened the pSB1A7 + RS1, and pSB1A7 + RS2 by PCR using the VF2 and RS1/RS2 Reverse Primers determine whether the plasmids obtained from the recombinant cells contain the riboswitch, and if so, if it inserted in the correct orientation.<br />
<br />
<br />
<br />
===August 21, 2008===<br />
====Roxanne====<br />
-Ran the PCR products on a 1% Agarose Gel at 100 V for 33 minutes. The gel was empty with the exception of primer dimers.<br />
<br />
====Nathan Puhl, Roxanne====<br />
-went over the SELEX protocol with HJ to determine the primers we will need to do this, and how exactly we plan on perfoming the evolution.<br />
<br />
-setup a restriction digest for pSB1A7 using XbaI and SpeI, ran overnight.<br />
<br />
===August 22, 2008===<br />
====Christa, Munima, Nathan Puhl, Roxanne====<br />
Objective: Run a gel to confirm that appropriate inserts were amplified from the PCRs and do a gel extraction of the inserts to prepare them for the biobrick format.<br />
<br />
-Could not obtain a picture of the gel (1% agarose) of the half of the digested pSB1A7 (15 uL x 3 wells) and <br />
the recently amplified <br />
CheZ gene (15 uL x 3 wells) because the camera would not turn on. <br />
The CheZ gene appeared at the correct size (~700 bp).<br />
-Did a gel extraction of pSB1A7 and CheZ from that gel with the Qiagen MiniElute Gel Extraction kit. <br />
Final volume of each was 10 uL.<br />
-Ran another 1% agarose gel to confirm that the gel extraction was successful. Ran 1 uL of each sample. <br />
Bands appeared at appropriate sizes. The concentrations of pSB1A7 <br />
and CheZ were estimated to be 25 ng/uL and 80 ng/uL, respectively.<br />
<br />
Next step: Digest pSB1A7 with antarctic phosphatase. A restriction digest will be performed at a later date on CheZ to prepare it for insertion.<br />
<br />
===August 23, 2008===<br />
====Nathan Puhl, Roxanne====<br />
-Digested pSB1A7 with Antartic Phosphatase<br />
<br />
-9 uL of cut pSB1A7<br />
-1.5 uL of 10x Antarctic Phosphatase Buffer<br />
-1 uL of Antarctic Phosphatase Enzyme<br />
-3.5 uL of water<br />
<br />
Allowed the Reaction to take place for 30 minutes to remove the 5` Phosphates from the pSB1A7 plasmid to <br />
prevent religation.<br />
<br />
-Ran the remainder of the pSB1A7 plasmid from August 22nd on 1 1% Agarose Gel at 100 v for 27 minutes.<br />
<br />
-Gel Extracted the plasmid DNA.<br />
<br />
-Purified the Phosphatase reaction to isolate the pSB1A7 DNA.<br />
<br />
-Ran a 1% gel to quantify the amount of plasmid DNA present.<br />
<br />
-Ligated RS1 and RS2 into the dephosphorylated pSB1A7 using T4 DNA Ligase.<br />
<br />
-1 uL of RS1 or RS2<br />
-4 uL of dephosphorylated pSB1A7<br />
-1 uL of 10X T4 DNA Ligase Buffer<br />
-0.33 uL T4 DNA Ligase Enzyme<br />
-3.67 uL water<br />
<br />
Reaction was allowed to go overnight<br />
<br />
===August 24, 2008===<br />
====Nathan Puhl====<br />
-Transformed DH5a cells with the RS1+pSB1A7 or RS2+pSB1A7 plasmid on semi-solid agar plates containing 100 ug/mLof amppicillin.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabAugustTeam:University of Lethbridge/Notebook/GeneralLabAugust2008-08-24T23:42:31Z<p>Nathan.puhl: /* Christa, Munima, Nathan Puhl, Roxanne */</p>
<hr />
<div>(Aug. 1- 7 belong in other August notebooks)<br />
===August 1, 2008===<br />
====Nathan Puhl, Roxanne====<br />
Riboswitch 20 uL PCR. Set up 4 reactions (25 uL for each total volume). 1 uL or 1/100 pTopp and 1 uL or H1/100 PCR from July 28, 2008. <br />
<br />
PCR conditions:<br />
A. Initial denaturation: 98 C (3 min)<br />
B. -Denaturation: 98 C (10 sec)<br />
- Annealing: 55 C (30 sec)<br />
-Extension: 72 C (15 sec)<br />
-30 cycles<br />
C. Final extension: 72 C (7 min)<br />
<br />
===August 2, 2008===<br />
====Nathan Puhl, Roxanne====<br />
Ran riboswitch (Aug. 1, 2008) on 3% agarose gel. Results are in the hard copy lab notebook. Looks like 76 bp band will extract.<br />
<br />
===August 5, 2008===<br />
====Nathan Puhl et al====<br />
Gel extracted two bands really closer to each other from PCR (August 1) at ~76 bp. Used Qiagen MiniElute kit. Results are in the hard copy lab notebook. Seems as if gel extraction was successful.<br />
____<br />
2 color reporter system:<br />
<br />
Set up digest of LacI and DT.<br />
<br />
LacI - EcoRI and SpeI<br />
<br />
DT - EcoRI and XbaI<br />
<br />
Reaction mixture:<br />
-5 uL template<br />
-5 uL NEB 2 Buffer<br />
-0.5 uL BSA?<br />
-1 uL RE #1<br />
-1 uL RE #2<br />
- 37.5 uL ddH20<br />
<br />
Left at 37 C overnight. Heat deactivation (65 C) for 15 minutes.<br />
<br />
<br />
===August 6, 2008===<br />
====Nathan Puhl, Roxanne====<br />
2 color reporter system:<br />
<br />
Gel extracted LacI insert and DT vector. Didn't run gel yet.<br />
<br />
===August 7, 2008===<br />
<br />
<br />
<br />
====Nathan Puhl, Roxanne====<br />
Riboswitch:<br />
<br />
Set up PCR using purified riboswitch from Aug. 5, 2008 with platinum Taq (50 uL reaction). Made Master Mix for three reactions.<br />
Master Mix:<br />
-10x Buffer (no Mg2+): 15 uL<br />
-10 mM dNTPs: 3 uL<br />
-50 mM Mg2+: 4.5 uL<br />
-10uM RF: 3 uL<br />
-10uM RR: 3 uL<br />
-Plat. poly: 0.6 iL<br />
-H20: 120.9 uL<br />
-template: 1 uL<br />
<br />
Cycle conditions:<br />
A. Initial denaturation: 94 C (2 min)<br />
B. -Denaturation: 94 C (30 sec)<br />
- Annealing: 55 C (30 sec)<br />
-Extension: 72 C (30 sec)<br />
-30 cycles<br />
C. Final extension: 72 C (7 min)<br />
<br />
====Roxanne====<br />
-Ran the PCR Product on a 3% gel using only 1uL of DNA from the riboswitch.<br />
<br />
===August 13, 2008===<br />
====Nathan Puhl and Sebastian====<br />
Verified and quantified the LacI and DT restriction digest products.<br />
<br />
Set up a Ligase experiment for LacI and DT<br />
- 1.3 uL vector(DT)<br />
- 8 uL insert(LacI)<br />
- 2 uL 10x Ligase buffer<br />
- 1.5 uL Ligase<br />
- 7.2 uL ddH2O<br />
- 20 uL total volume<br />
<br />
<br />
===August 14, 15===<br />
====Nathan Puhl, Munima, Selina====<br />
Poured 61 LB + Amp plates. Stored in iGEM 4 C fridge.<br />
<br />
<br />
===August 15, 2008===<br />
====Roxanne====<br />
<br />
Used Qiagen Plasmid MiniPrep Kit to Plasmid Prep Last Year's Biobrick Parts that were Incubated overnight.<br />
<br />
Ran Plasmids in a 1% Agarose Gel at 100 V for 30 minutes.<br />
<br />
===August 16, 2008===<br />
<br />
====Nathan Puhl, Roxanne, Munima====<br />
-Restriction Digested the purified riboswitch and pSB1A7 with XbaI and SpeI, let it run for 4 hours<br />
<br />
<br />
====Nathan Puhl, Roxanne====<br />
-Ran all of the restricted pSB1A7 plasmid through a 1% agarose gel at 100V for 25 minutes.<br />
-Ran a gel extraction on the pSB1A7 cut plasmid, and ran a PCR clean-up reaction on the digested RS1 and RS2 amplicons.<br />
-Ran 1 uL of each on a 1% to quantify the amount of DNA present.<br />
-Ligated RS1 + pSB1A7, and RS2 + pSB1A7, using T4 DNA Ligase.<br />
<br />
-1 uL of RS1/RS2<br />
-4 uL of pSB1A7<br />
-1 uL of 10x T4 DNA Ligase Buffer<br />
-0.33 uL of T4 DNA Ligase<br />
-3.67 uL of water<br />
<br />
allowed the reaction to go overnight<br />
<br />
<br />
===August 17, 2008===<br />
====Nathan Puhl====<br />
-Transformed DH5a cells with the pSB1A7 + RS1, and pSB1A7 + RS2 plasmids.<br />
<br />
-Plated on semi-solid agar plates containing 100 ug/mL of ampicillin.<br />
<br />
===August 18===<br />
====Christa, Nathan Puhl, Munima====<br />
-Nathan checked the plates for growth. Colonies are present.<br />
<br />
-Ran a colony PCR of the pSB1A7 + RS1, and pSB1A7 + RS2 recombinant cells transformed by Nathan and Roxanne on August 17.<br />
<br />
-Inoculated the cells into tubes of liquid media + 100 ug/ml of ampicillin.<br />
<br />
Roxanne will remove the PCR tube from the thermocycler in the morning.<br />
<br />
===August 19, 2008===<br />
<br />
====Roxanne====<br />
-Ran the PCR Product on a 2% Agarose Gel at 100 V for 33 minutes.<br />
<br />
[[Image:RS1 RS2 gel.jpg| 350 px]]<br />
<br />
-Plasmid Prepped and made glycerol stocks from the RS1-1 and RS2-1 tubes of cells incubated in LB media + 100 ug/mL ampicillin.<br />
<br />
Ran the pRS1 and pRS2 plasmids on a 1% Agarose Gel at 100 V for 30 minutes.<br />
<br />
[[Image:pRS1 pRS2 gel.jpg| 150 px]]<br />
<br />
====Christa, Munima, Sebastian====<br />
Did a plasmid prep on pSB1A7 using QIAprep Spin Miniprep Kit. Stocked 4- 50uL of pSB1A7 and stored in iGEM -20 C.<br />
<br />
====Sebastian, Nathan Puhl====<br />
Ran products from plasmid prep (pSB1A7 x 4 samples) on 1% agarose gel for 27 minutes.<br />
<br />
-Lane 1 - 1 kb GeneRuler ladder (2 uL)<br />
-Lane 2 -6 pSB1A7 (3 uL) + 6x loading dye (2 uL); Mixed up what sample was in Lane 4, so Lanes 5 and 6 were run.<br />
<br />
Conclusion: The bands appeared to be at the correct size for pSB1A7.<br />
<br />
===August 21===<br />
====Nathan Puhl, Roxanne====<br />
-Screened the pSB1A7 + RS1, and pSB1A7 + RS2 by PCR using the VF2 and RS1/RS2 Reverse Primers determine whether the plasmids obtained from the recombinant cells contain the riboswitch, and if so, if it inserted in the correct orientation.<br />
<br />
<br />
<br />
===August 21, 2008===<br />
====Roxanne====<br />
-Ran the PCR products on a 1% Agarose Gel at 100 V for 33 minutes. The gel was empty with the exception of primer dimers.<br />
<br />
====Nathan Puhl, Roxanne====<br />
-went over the SELEX protocol with HJ to determine the primers we will need to do this, and how exactly we plan on perfoming the evolution.<br />
<br />
-setup a restriction digest for pSB1A7 using XbaI and SpeI, ran overnight.<br />
<br />
===August 22, 2008===<br />
====Christa, Munima, Nathan Puhl, Roxanne====<br />
Objective: Run a gel to confirm that appropriate inserts were amplified from the PCRs and do a gel extraction of the inserts to prepare them for the biobrick format.<br />
<br />
-Could not obtain a picture of the gel (1% agarose) of the half of the digested pSB1A7 (15 uL x 3 wells) and <br />
the recently amplified <br />
CheZ gene (15 uL x 3 wells) because the camera would not turn on. <br />
The CheZ gene appeared at the correct size (~700 bp).<br />
-Did a gel extraction of pSB1A7 and CheZ from that gel with the Qiagen MiniElute Gel Extraction kit. <br />
Final volume of each was 10 uL.<br />
-Ran another 1% agarose gel to confirm that the gel extraction was successful. Ran 1 uL of each sample. <br />
Bands appeared at appropriate sizes. The concentrations of pSB1A7 <br />
and CheZ were estimated to be 25 ng/uL and 80 ng/uL, respectively.<br />
<br />
Next step: Digest pSB1A7 with alkaline phosphatase. A restriction digest will be performed at a later date on CheZ to prepare it for insertion.<br />
<br />
===August 23, 2008===<br />
====Nathan Puhl, Roxanne====<br />
-Digested pSB1A7 with Antartic Phosphatase<br />
<br />
-9 uL of cut pSB1A7<br />
-1.5 uL of 10x Antarctic Phosphatase Buffer<br />
-1 uL of Antarctic Phosphatase Enzyme<br />
-3.5 uL of water<br />
<br />
Allowed the Reaction to take place for 30 minutes to remove the 5` Phosphates from the pSB1A7 plasmid to <br />
prevent religation.<br />
<br />
-Ran the remainder of the pSB1A7 plasmid from August 22nd on 1 1% Agarose Gel at 100 v for 27 minutes.<br />
<br />
-Gel Extracted the plasmid DNA.<br />
<br />
-Purified the Phosphatase reaction to isolate the pSB1A7 DNA.<br />
<br />
-Ran a 1% gel to quantify the amount of plasmid DNA present.<br />
<br />
-Ligated RS1 and RS2 into the dephosphorylated pSB1A7 using T4 DNA Ligase.<br />
<br />
-1 uL of RS1 or RS2<br />
-4 uL of dephosphorylated pSB1A7<br />
-1 uL of 10X T4 DNA Ligase Buffer<br />
-0.33 uL T4 DNA Ligase Enzyme<br />
-3.67 uL water<br />
<br />
Reaction was allowed to go overnight<br />
<br />
===August 24, 2008===<br />
====Nathan Puhl====<br />
-Transformed DH5a cells with the RS1+pSB1A7 or RS2+pSB1A7 plasmid on semi-solid agar plates containing 100 ug/mLof amppicillin.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabAugustTeam:University of Lethbridge/Notebook/GeneralLabAugust2008-08-24T23:42:11Z<p>Nathan.puhl: /* Christa, Munima, Nathan Puhl, Roxanne */</p>
<hr />
<div>(Aug. 1- 7 belong in other August notebooks)<br />
===August 1, 2008===<br />
====Nathan Puhl, Roxanne====<br />
Riboswitch 20 uL PCR. Set up 4 reactions (25 uL for each total volume). 1 uL or 1/100 pTopp and 1 uL or H1/100 PCR from July 28, 2008. <br />
<br />
PCR conditions:<br />
A. Initial denaturation: 98 C (3 min)<br />
B. -Denaturation: 98 C (10 sec)<br />
- Annealing: 55 C (30 sec)<br />
-Extension: 72 C (15 sec)<br />
-30 cycles<br />
C. Final extension: 72 C (7 min)<br />
<br />
===August 2, 2008===<br />
====Nathan Puhl, Roxanne====<br />
Ran riboswitch (Aug. 1, 2008) on 3% agarose gel. Results are in the hard copy lab notebook. Looks like 76 bp band will extract.<br />
<br />
===August 5, 2008===<br />
====Nathan Puhl et al====<br />
Gel extracted two bands really closer to each other from PCR (August 1) at ~76 bp. Used Qiagen MiniElute kit. Results are in the hard copy lab notebook. Seems as if gel extraction was successful.<br />
____<br />
2 color reporter system:<br />
<br />
Set up digest of LacI and DT.<br />
<br />
LacI - EcoRI and SpeI<br />
<br />
DT - EcoRI and XbaI<br />
<br />
Reaction mixture:<br />
-5 uL template<br />
-5 uL NEB 2 Buffer<br />
-0.5 uL BSA?<br />
-1 uL RE #1<br />
-1 uL RE #2<br />
- 37.5 uL ddH20<br />
<br />
Left at 37 C overnight. Heat deactivation (65 C) for 15 minutes.<br />
<br />
<br />
===August 6, 2008===<br />
====Nathan Puhl, Roxanne====<br />
2 color reporter system:<br />
<br />
Gel extracted LacI insert and DT vector. Didn't run gel yet.<br />
<br />
===August 7, 2008===<br />
<br />
<br />
<br />
====Nathan Puhl, Roxanne====<br />
Riboswitch:<br />
<br />
Set up PCR using purified riboswitch from Aug. 5, 2008 with platinum Taq (50 uL reaction). Made Master Mix for three reactions.<br />
Master Mix:<br />
-10x Buffer (no Mg2+): 15 uL<br />
-10 mM dNTPs: 3 uL<br />
-50 mM Mg2+: 4.5 uL<br />
-10uM RF: 3 uL<br />
-10uM RR: 3 uL<br />
-Plat. poly: 0.6 iL<br />
-H20: 120.9 uL<br />
-template: 1 uL<br />
<br />
Cycle conditions:<br />
A. Initial denaturation: 94 C (2 min)<br />
B. -Denaturation: 94 C (30 sec)<br />
- Annealing: 55 C (30 sec)<br />
-Extension: 72 C (30 sec)<br />
-30 cycles<br />
C. Final extension: 72 C (7 min)<br />
<br />
====Roxanne====<br />
-Ran the PCR Product on a 3% gel using only 1uL of DNA from the riboswitch.<br />
<br />
===August 13, 2008===<br />
====Nathan Puhl and Sebastian====<br />
Verified and quantified the LacI and DT restriction digest products.<br />
<br />
Set up a Ligase experiment for LacI and DT<br />
- 1.3 uL vector(DT)<br />
- 8 uL insert(LacI)<br />
- 2 uL 10x Ligase buffer<br />
- 1.5 uL Ligase<br />
- 7.2 uL ddH2O<br />
- 20 uL total volume<br />
<br />
<br />
===August 14, 15===<br />
====Nathan Puhl, Munima, Selina====<br />
Poured 61 LB + Amp plates. Stored in iGEM 4 C fridge.<br />
<br />
<br />
===August 15, 2008===<br />
====Roxanne====<br />
<br />
Used Qiagen Plasmid MiniPrep Kit to Plasmid Prep Last Year's Biobrick Parts that were Incubated overnight.<br />
<br />
Ran Plasmids in a 1% Agarose Gel at 100 V for 30 minutes.<br />
<br />
===August 16, 2008===<br />
<br />
====Nathan Puhl, Roxanne, Munima====<br />
-Restriction Digested the purified riboswitch and pSB1A7 with XbaI and SpeI, let it run for 4 hours<br />
<br />
<br />
====Nathan Puhl, Roxanne====<br />
-Ran all of the restricted pSB1A7 plasmid through a 1% agarose gel at 100V for 25 minutes.<br />
-Ran a gel extraction on the pSB1A7 cut plasmid, and ran a PCR clean-up reaction on the digested RS1 and RS2 amplicons.<br />
-Ran 1 uL of each on a 1% to quantify the amount of DNA present.<br />
-Ligated RS1 + pSB1A7, and RS2 + pSB1A7, using T4 DNA Ligase.<br />
<br />
-1 uL of RS1/RS2<br />
-4 uL of pSB1A7<br />
-1 uL of 10x T4 DNA Ligase Buffer<br />
-0.33 uL of T4 DNA Ligase<br />
-3.67 uL of water<br />
<br />
allowed the reaction to go overnight<br />
<br />
<br />
===August 17, 2008===<br />
====Nathan Puhl====<br />
-Transformed DH5a cells with the pSB1A7 + RS1, and pSB1A7 + RS2 plasmids.<br />
<br />
-Plated on semi-solid agar plates containing 100 ug/mL of ampicillin.<br />
<br />
===August 18===<br />
====Christa, Nathan Puhl, Munima====<br />
-Nathan checked the plates for growth. Colonies are present.<br />
<br />
-Ran a colony PCR of the pSB1A7 + RS1, and pSB1A7 + RS2 recombinant cells transformed by Nathan and Roxanne on August 17.<br />
<br />
-Inoculated the cells into tubes of liquid media + 100 ug/ml of ampicillin.<br />
<br />
Roxanne will remove the PCR tube from the thermocycler in the morning.<br />
<br />
===August 19, 2008===<br />
<br />
====Roxanne====<br />
-Ran the PCR Product on a 2% Agarose Gel at 100 V for 33 minutes.<br />
<br />
[[Image:RS1 RS2 gel.jpg| 350 px]]<br />
<br />
-Plasmid Prepped and made glycerol stocks from the RS1-1 and RS2-1 tubes of cells incubated in LB media + 100 ug/mL ampicillin.<br />
<br />
Ran the pRS1 and pRS2 plasmids on a 1% Agarose Gel at 100 V for 30 minutes.<br />
<br />
[[Image:pRS1 pRS2 gel.jpg| 150 px]]<br />
<br />
====Christa, Munima, Sebastian====<br />
Did a plasmid prep on pSB1A7 using QIAprep Spin Miniprep Kit. Stocked 4- 50uL of pSB1A7 and stored in iGEM -20 C.<br />
<br />
====Sebastian, Nathan Puhl====<br />
Ran products from plasmid prep (pSB1A7 x 4 samples) on 1% agarose gel for 27 minutes.<br />
<br />
-Lane 1 - 1 kb GeneRuler ladder (2 uL)<br />
-Lane 2 -6 pSB1A7 (3 uL) + 6x loading dye (2 uL); Mixed up what sample was in Lane 4, so Lanes 5 and 6 were run.<br />
<br />
Conclusion: The bands appeared to be at the correct size for pSB1A7.<br />
<br />
===August 21===<br />
====Nathan Puhl, Roxanne====<br />
-Screened the pSB1A7 + RS1, and pSB1A7 + RS2 by PCR using the VF2 and RS1/RS2 Reverse Primers determine whether the plasmids obtained from the recombinant cells contain the riboswitch, and if so, if it inserted in the correct orientation.<br />
<br />
<br />
<br />
===August 21, 2008===<br />
====Roxanne====<br />
-Ran the PCR products on a 1% Agarose Gel at 100 V for 33 minutes. The gel was empty with the exception of primer dimers.<br />
<br />
====Nathan Puhl, Roxanne====<br />
-went over the SELEX protocol with HJ to determine the primers we will need to do this, and how exactly we plan on perfoming the evolution.<br />
<br />
-setup a restriction digest for pSB1A7 using XbaI and SpeI, ran overnight.<br />
<br />
===August 22, 2008===<br />
====Christa, Munima, Nathan Puhl, Roxanne====<br />
Objective: Run a gel to confirm that appropriate inserts were amplified from the PCRs and do a gel extraction of the inserts to prepare them for the biobrick format.<br />
<br />
-Could not obtain a picture of the gel (1% agarose) of the half of the digested pSB1A7 (15 uL x 3 wells) and <br />
the recently amplified <br />
CheZ gene (15 uL x 3 wells) because the camera would not turn on. <br />
The CheZ gene appeared at the correct size (~700 bp).<br />
-Did a gel extraction of pSB1A7 and CheZ from that gel with the Qiagen MiniElute Gel Extraction kit. <br />
Final volume of each was 10 uL.<br />
-Ran another 1% agarose gel to confirm that the gel extraction was successful. Ran 1 uL of each sample. <br />
Bands appeared at appropriate sizes. The concentrations of pSB1A7 <br />
and CheZ were estimated to be 25 ng/uL and 80 ng/uL, respectively.<br />
<br />
Next step: Digest pSB1A7 and cheZ with alkaline phosphatase. A restriction digest will be performed at a later date on CheZ to prepare it for insertion.<br />
<br />
===August 23, 2008===<br />
====Nathan Puhl, Roxanne====<br />
-Digested pSB1A7 with Antartic Phosphatase<br />
<br />
-9 uL of cut pSB1A7<br />
-1.5 uL of 10x Antarctic Phosphatase Buffer<br />
-1 uL of Antarctic Phosphatase Enzyme<br />
-3.5 uL of water<br />
<br />
Allowed the Reaction to take place for 30 minutes to remove the 5` Phosphates from the pSB1A7 plasmid to <br />
prevent religation.<br />
<br />
-Ran the remainder of the pSB1A7 plasmid from August 22nd on 1 1% Agarose Gel at 100 v for 27 minutes.<br />
<br />
-Gel Extracted the plasmid DNA.<br />
<br />
-Purified the Phosphatase reaction to isolate the pSB1A7 DNA.<br />
<br />
-Ran a 1% gel to quantify the amount of plasmid DNA present.<br />
<br />
-Ligated RS1 and RS2 into the dephosphorylated pSB1A7 using T4 DNA Ligase.<br />
<br />
-1 uL of RS1 or RS2<br />
-4 uL of dephosphorylated pSB1A7<br />
-1 uL of 10X T4 DNA Ligase Buffer<br />
-0.33 uL T4 DNA Ligase Enzyme<br />
-3.67 uL water<br />
<br />
Reaction was allowed to go overnight<br />
<br />
===August 24, 2008===<br />
====Nathan Puhl====<br />
-Transformed DH5a cells with the RS1+pSB1A7 or RS2+pSB1A7 plasmid on semi-solid agar plates containing 100 ug/mLof amppicillin.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabAugstTeam:University of Lethbridge/Notebook/GeneralLabAugst2008-08-08T01:41:25Z<p>Nathan.puhl: </p>
<hr />
<div>===August 01, 2008===<br />
====Nathan Puhl, Nathan Phillips====<br />
<br />
'''Objective:''' Amplify more of the riboswitch using amplicons from the previous PCR<br />
<br />
MasterMix:<br />
1x 4x <br />
5x buffer 5 uL 20 <br />
10 mM dNTP 0.5 uL 2 <br />
RF primer 2.5 uL 10 <br />
RR primer 2.5 uL 10 <br />
Phusion 0.25 uL 1 <br />
ddH2O 15.25 uL 61<br />
Template 1 uL of pTOPP, 1x, 1/10x riboswitch amplicon<br />
<br />
Cycle Conditions:<br />
98 C - 3 min<br />
30x: 98 C - 10 sec<br />
55 C - 30 sec<br />
72 C - 15 sec<br />
72 C - 7 min<br />
<br />
===August 02, 2008===<br />
====Nathan Puhl====<br />
Ran gel of PCR products from Aug. 01 with pTOPP template in lane 2, 1x, 1/10x amplicon template in lane 3 and 4<br />
<br />
[[Image:riboswitch-1.jpg]]<br />
<br />
===August 03, 2008===<br />
====Nathan Phillips====<br />
<br />
Tranformation of pUC19:<br />
1. Thaw E.coli DH5a cells on ice<br />
2. Add pUC19 DNA, pipette gently to mix (1μl of plasmid)<br />
3. Let sit for 30 minutes on ice<br />
4. Incubate cells for 30 seconds at 42oC<br />
5. Incubate cells on ice for 2 min<br />
6. Add 1 mL SOC at room temp<br />
7. Incubate for 1 hour at 37oC on shaker<br />
8. Spread 100-300 μl onto a plate made with appropriate antibiotic<br />
9. Grow overnight at 37 °C<br />
<br />
===August 5, 2008===<br />
====Nathan Puhl, Alix, Roxanne and highschool students====<br />
'''Objective:''' Isolate 76bp band from non-specific amplified bands of riboswitch PCR<br />
<br />
Using Qiagen MinElute Kit extracted two very, very similiarly sized bands after running on a 3.5% agarose gel.<br />
<br />
[[Image:purified riboswitch.jpg]]<br />
<br />
'''Objective:''' Cut out LacI gene and open DT vector for construction<br />
<br />
Restriction Digest of BBa_C0012 (LacI) and BBa_C0012 (Double Terminator)<br />
<br />
LacI - EcoR I + Spe I<br />
<br />
DT - EcoR I + Xba I<br />
<br />
Overnight incubation at 37 C followed by 65 C for 15 min (enzyme deactivation)<br />
<br />
Reaction Mixture: <br />
-5 uL template <br />
-5 uL NEB 2 buffer<br />
-0.5 uL BSA<br />
-1 uL restriction enzyme #1<br />
-1 uL restriction enzyme #2<br />
-37.5 uL ddH2O<br />
<br />
===August 6, 2008===<br />
====Nathan Puhl, Roxanne, Alix====<br />
<br />
'''Objective:''' Gel extract digested biobricks to clean for ligation reaction<br />
<br />
Qiagen MinElute Kit was used to gel purify the digested LacI and DT biobricks.<br />
<br />
===August 7, 2008===<br />
====Nathan Puhl, Roxanne, Peter====<br />
'''Objective:''' Amplify riboswitch with A overhangs for TA cloning<br />
<br />
Set up PCR using purified riboswitch amplicon from Aug. 05 (1 uL of template)<br />
<br />
MasterMix:<br />
1x 3x <br />
10x buffer 5 uL 15 <br />
10 mM dNTP 1 uL 3 <br />
50 mM Mg2+ 1.5 4.5<br />
RF primer 1 uL 3 <br />
RR primer 1 uL 3 <br />
Platinum taq 0.2 uL 0.6 <br />
ddH2O 39.3 uL 117.9<br />
Template 1 uL of riboswitch amplicon<br />
<br />
Cycle Conditions:<br />
94 C - 2 min<br />
30x: 94 C - 30 sec<br />
53 C - 30 sec<br />
72 C - 30 sec</div>Nathan.puhlhttp://2008.igem.org/File:Riboswitch-1.jpgFile:Riboswitch-1.jpg2008-08-08T01:35:36Z<p>Nathan.puhl: </p>
<hr />
<div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabAugstTeam:University of Lethbridge/Notebook/GeneralLabAugst2008-08-08T01:35:20Z<p>Nathan.puhl: /* August 02, 2008 */</p>
<hr />
<div>===August 01, 2008===<br />
====Nathan Puhl, Nathan Phillips====<br />
<br />
'''Objective:''' Amplify more of the riboswitch using amplicons from the previous PCR<br />
<br />
MasterMix:<br />
1x 4x <br />
5x buffer 5 uL 20 <br />
10 mM dNTP 0.5 uL 2 <br />
RF primer 2.5 uL 10 <br />
RR primer 2.5 uL 10 <br />
Phusion 0.25 uL 1 <br />
ddH2O 15.25 uL 61<br />
Template 1 uL of pTOPP, 1x, 1/10x riboswitch amplicon<br />
<br />
Cycle Conditions:<br />
98 C - 3 min<br />
30x: 98 C - 10 sec<br />
55 C - 30 sec<br />
72 C - 15 sec<br />
72 C - 7 min<br />
<br />
===August 02, 2008===<br />
====Nathan Puhl====<br />
Ran gel of PCR products from Aug. 01 with pTOPP template in lane 2, 1x, 1/10x amplicon template in lane 3 and 4<br />
<br />
[[Image:riboswitch-1.jpg]]<br />
<br />
===August 03, 2008===<br />
====Nathan Phillips====<br />
<br />
Tranformation of pUC19:<br />
1. Thaw E.coli DH5a cells on ice<br />
2. Add pUC19 DNA, pipette gently to mix (1μl of plasmid)<br />
3. Let sit for 30 minutes on ice<br />
4. Incubate cells for 30 seconds at 42oC<br />
5. Incubate cells on ice for 2 min<br />
6. Add 1 mL SOC at room temp<br />
7. Incubate for 1 hour at 37oC on shaker<br />
8. Spread 100-300 μl onto a plate made with appropriate antibiotic<br />
9. Grow overnight at 37 °C<br />
<br />
===August 5, 2008===<br />
====Nathan Puhl, Alix, Roxanne and highschool students====<br />
'''Objective:''' Isolate 76bp band from non-specific amplified bands of riboswitch PCR<br />
<br />
Using Qiagen MinElute Kit extracted two very, very similiarly sized bands after running on a 3.5% agarose gel.<br />
<br />
[[Image:purified riboswitch.jpg]]<br />
<br />
'''Objective:''' Cut out LacI gene and open DT vector for construction<br />
<br />
Restriction Digest of BBa_C0012 (LacI) and BBa_C0012 (Double Terminator)<br />
<br />
LacI - EcoR I + Spe I<br />
<br />
DT - EcoR I + Xba I<br />
<br />
Overnight incubation at 37 C followed by 65 C for 15 min (enzyme deactivation)<br />
<br />
Reaction Mixture: <br />
-5 uL template <br />
-5 uL NEB 2 buffer<br />
-0.5 uL BSA<br />
-1 uL restriction enzyme #1<br />
-1 uL restriction enzyme #2<br />
-37.5 uL ddH2O<br />
<br />
===August 6, 2008===<br />
====Nathan Puhl, Roxanne, Alix====<br />
<br />
'''Objective:''' Gel extract digested biobricks to clean for ligation reaction<br />
<br />
Qiagen MinElute Kit was used to gel purify the digested LacI and DT biobricks.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabAugstTeam:University of Lethbridge/Notebook/GeneralLabAugst2008-08-08T01:35:01Z<p>Nathan.puhl: /* =August 02, 2008 */</p>
<hr />
<div>===August 01, 2008===<br />
====Nathan Puhl, Nathan Phillips====<br />
<br />
'''Objective:''' Amplify more of the riboswitch using amplicons from the previous PCR<br />
<br />
MasterMix:<br />
1x 4x <br />
5x buffer 5 uL 20 <br />
10 mM dNTP 0.5 uL 2 <br />
RF primer 2.5 uL 10 <br />
RR primer 2.5 uL 10 <br />
Phusion 0.25 uL 1 <br />
ddH2O 15.25 uL 61<br />
Template 1 uL of pTOPP, 1x, 1/10x riboswitch amplicon<br />
<br />
Cycle Conditions:<br />
98 C - 3 min<br />
30x: 98 C - 10 sec<br />
55 C - 30 sec<br />
72 C - 15 sec<br />
72 C - 7 min<br />
<br />
===August 02, 2008===<br />
====Nathan Puhl=====<br />
Ran gel of PCR products from Aug. 01 with pTOPP template in lane 2, 1x, 1/10x amplicon template in lane 3 and 4<br />
<br />
[[Image:riboswitch-1.jpg]]<br />
<br />
===August 03, 2008===<br />
====Nathan Phillips====<br />
<br />
Tranformation of pUC19:<br />
1. Thaw E.coli DH5a cells on ice<br />
2. Add pUC19 DNA, pipette gently to mix (1μl of plasmid)<br />
3. Let sit for 30 minutes on ice<br />
4. Incubate cells for 30 seconds at 42oC<br />
5. Incubate cells on ice for 2 min<br />
6. Add 1 mL SOC at room temp<br />
7. Incubate for 1 hour at 37oC on shaker<br />
8. Spread 100-300 μl onto a plate made with appropriate antibiotic<br />
9. Grow overnight at 37 °C<br />
<br />
===August 5, 2008===<br />
====Nathan Puhl, Alix, Roxanne and highschool students====<br />
'''Objective:''' Isolate 76bp band from non-specific amplified bands of riboswitch PCR<br />
<br />
Using Qiagen MinElute Kit extracted two very, very similiarly sized bands after running on a 3.5% agarose gel.<br />
<br />
[[Image:purified riboswitch.jpg]]<br />
<br />
'''Objective:''' Cut out LacI gene and open DT vector for construction<br />
<br />
Restriction Digest of BBa_C0012 (LacI) and BBa_C0012 (Double Terminator)<br />
<br />
LacI - EcoR I + Spe I<br />
<br />
DT - EcoR I + Xba I<br />
<br />
Overnight incubation at 37 C followed by 65 C for 15 min (enzyme deactivation)<br />
<br />
Reaction Mixture: <br />
-5 uL template <br />
-5 uL NEB 2 buffer<br />
-0.5 uL BSA<br />
-1 uL restriction enzyme #1<br />
-1 uL restriction enzyme #2<br />
-37.5 uL ddH2O<br />
<br />
===August 6, 2008===<br />
====Nathan Puhl, Roxanne, Alix====<br />
<br />
'''Objective:''' Gel extract digested biobricks to clean for ligation reaction<br />
<br />
Qiagen MinElute Kit was used to gel purify the digested LacI and DT biobricks.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabAugstTeam:University of Lethbridge/Notebook/GeneralLabAugst2008-08-08T01:34:46Z<p>Nathan.puhl: /* Nathan Puhl= */</p>
<hr />
<div>===August 01, 2008===<br />
====Nathan Puhl, Nathan Phillips====<br />
<br />
'''Objective:''' Amplify more of the riboswitch using amplicons from the previous PCR<br />
<br />
MasterMix:<br />
1x 4x <br />
5x buffer 5 uL 20 <br />
10 mM dNTP 0.5 uL 2 <br />
RF primer 2.5 uL 10 <br />
RR primer 2.5 uL 10 <br />
Phusion 0.25 uL 1 <br />
ddH2O 15.25 uL 61<br />
Template 1 uL of pTOPP, 1x, 1/10x riboswitch amplicon<br />
<br />
Cycle Conditions:<br />
98 C - 3 min<br />
30x: 98 C - 10 sec<br />
55 C - 30 sec<br />
72 C - 15 sec<br />
72 C - 7 min<br />
<br />
===August 02, 2008==<br />
====Nathan Puhl=====<br />
Ran gel of PCR products from Aug. 01 with pTOPP template in lane 2, 1x, 1/10x amplicon template in lane 3 and 4<br />
<br />
[[Image:riboswitch-1.jpg]]<br />
<br />
===August 03, 2008===<br />
====Nathan Phillips====<br />
<br />
Tranformation of pUC19:<br />
1. Thaw E.coli DH5a cells on ice<br />
2. Add pUC19 DNA, pipette gently to mix (1μl of plasmid)<br />
3. Let sit for 30 minutes on ice<br />
4. Incubate cells for 30 seconds at 42oC<br />
5. Incubate cells on ice for 2 min<br />
6. Add 1 mL SOC at room temp<br />
7. Incubate for 1 hour at 37oC on shaker<br />
8. Spread 100-300 μl onto a plate made with appropriate antibiotic<br />
9. Grow overnight at 37 °C<br />
<br />
===August 5, 2008===<br />
====Nathan Puhl, Alix, Roxanne and highschool students====<br />
'''Objective:''' Isolate 76bp band from non-specific amplified bands of riboswitch PCR<br />
<br />
Using Qiagen MinElute Kit extracted two very, very similiarly sized bands after running on a 3.5% agarose gel.<br />
<br />
[[Image:purified riboswitch.jpg]]<br />
<br />
'''Objective:''' Cut out LacI gene and open DT vector for construction<br />
<br />
Restriction Digest of BBa_C0012 (LacI) and BBa_C0012 (Double Terminator)<br />
<br />
LacI - EcoR I + Spe I<br />
<br />
DT - EcoR I + Xba I<br />
<br />
Overnight incubation at 37 C followed by 65 C for 15 min (enzyme deactivation)<br />
<br />
Reaction Mixture: <br />
-5 uL template <br />
-5 uL NEB 2 buffer<br />
-0.5 uL BSA<br />
-1 uL restriction enzyme #1<br />
-1 uL restriction enzyme #2<br />
-37.5 uL ddH2O<br />
<br />
===August 6, 2008===<br />
====Nathan Puhl, Roxanne, Alix====<br />
<br />
'''Objective:''' Gel extract digested biobricks to clean for ligation reaction<br />
<br />
Qiagen MinElute Kit was used to gel purify the digested LacI and DT biobricks.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabAugstTeam:University of Lethbridge/Notebook/GeneralLabAugst2008-08-08T01:34:26Z<p>Nathan.puhl: </p>
<hr />
<div>===August 01, 2008===<br />
====Nathan Puhl, Nathan Phillips====<br />
<br />
'''Objective:''' Amplify more of the riboswitch using amplicons from the previous PCR<br />
<br />
MasterMix:<br />
1x 4x <br />
5x buffer 5 uL 20 <br />
10 mM dNTP 0.5 uL 2 <br />
RF primer 2.5 uL 10 <br />
RR primer 2.5 uL 10 <br />
Phusion 0.25 uL 1 <br />
ddH2O 15.25 uL 61<br />
Template 1 uL of pTOPP, 1x, 1/10x riboswitch amplicon<br />
<br />
Cycle Conditions:<br />
98 C - 3 min<br />
30x: 98 C - 10 sec<br />
55 C - 30 sec<br />
72 C - 15 sec<br />
72 C - 7 min<br />
<br />
===August 02, 2008==<br />
====Nathan Puhl=====<br />
Ran gel of PCR products from Aug. 01 with pTOPP template in lane 2, 1x, 1/10x amplicon template in lane 3 and 4<br />
<br />
[[Image:riboswitch.jpg]]<br />
<br />
===August 03, 2008===<br />
====Nathan Phillips====<br />
<br />
Tranformation of pUC19:<br />
1. Thaw E.coli DH5a cells on ice<br />
2. Add pUC19 DNA, pipette gently to mix (1μl of plasmid)<br />
3. Let sit for 30 minutes on ice<br />
4. Incubate cells for 30 seconds at 42oC<br />
5. Incubate cells on ice for 2 min<br />
6. Add 1 mL SOC at room temp<br />
7. Incubate for 1 hour at 37oC on shaker<br />
8. Spread 100-300 μl onto a plate made with appropriate antibiotic<br />
9. Grow overnight at 37 °C<br />
<br />
===August 5, 2008===<br />
====Nathan Puhl, Alix, Roxanne and highschool students====<br />
'''Objective:''' Isolate 76bp band from non-specific amplified bands of riboswitch PCR<br />
<br />
Using Qiagen MinElute Kit extracted two very, very similiarly sized bands after running on a 3.5% agarose gel.<br />
<br />
[[Image:purified riboswitch.jpg]]<br />
<br />
'''Objective:''' Cut out LacI gene and open DT vector for construction<br />
<br />
Restriction Digest of BBa_C0012 (LacI) and BBa_C0012 (Double Terminator)<br />
<br />
LacI - EcoR I + Spe I<br />
<br />
DT - EcoR I + Xba I<br />
<br />
Overnight incubation at 37 C followed by 65 C for 15 min (enzyme deactivation)<br />
<br />
Reaction Mixture: <br />
-5 uL template <br />
-5 uL NEB 2 buffer<br />
-0.5 uL BSA<br />
-1 uL restriction enzyme #1<br />
-1 uL restriction enzyme #2<br />
-37.5 uL ddH2O<br />
<br />
===August 6, 2008===<br />
====Nathan Puhl, Roxanne, Alix====<br />
<br />
'''Objective:''' Gel extract digested biobricks to clean for ligation reaction<br />
<br />
Qiagen MinElute Kit was used to gel purify the digested LacI and DT biobricks.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabAugstTeam:University of Lethbridge/Notebook/GeneralLabAugst2008-08-08T01:32:02Z<p>Nathan.puhl: </p>
<hr />
<div>===August 01, 2008===<br />
====Nathan Puhl, Nathan Phillips====<br />
<br />
'''Objective:''' Amplify more of the riboswitch using amplicons from the previous PCR<br />
<br />
MasterMix:<br />
1x 4x <br />
5x buffer 5 uL 20 <br />
10 mM dNTP 0.5 uL 2 <br />
RF primer 2.5 uL 10 <br />
RR primer 2.5 uL 10 <br />
Phusion 0.25 uL 1 <br />
ddH2O 15.25 uL 61<br />
Template 1 uL of pTOPP, 1x, 1/10x riboswitch amplicon<br />
<br />
Cycle Conditions:<br />
98 C - 3 min<br />
30x: 98 C - 10 sec<br />
55 C - 30 sec<br />
72 C - 15 sec<br />
72 C - 7 min<br />
<br />
===August 03, 2008===<br />
====Nathan Phillips====<br />
<br />
Tranformation of pUC19:<br />
1. Thaw E.coli DH5a cells on ice<br />
2. Add pUC19 DNA, pipette gently to mix (1μl of plasmid)<br />
3. Let sit for 30 minutes on ice<br />
4. Incubate cells for 30 seconds at 42oC<br />
5. Incubate cells on ice for 2 min<br />
6. Add 1 mL SOC at room temp<br />
7. Incubate for 1 hour at 37oC on shaker<br />
8. Spread 100-300 μl onto a plate made with appropriate antibiotic<br />
9. Grow overnight at 37 °C<br />
<br />
===August 5, 2008===<br />
====Nathan Puhl, Alix, Roxanne and highschool students====<br />
'''Objective:''' Isolate 76bp band from non-specific amplified bands of riboswitch PCR<br />
<br />
Using Qiagen MinElute Kit extracted two very, very similiarly sized bands after running on a 3.5% agarose gel.<br />
<br />
[[Image:purified riboswitch.jpg]]<br />
<br />
'''Objective:''' Cut out LacI gene and open DT vector for construction<br />
<br />
Restriction Digest of BBa_C0012 (LacI) and BBa_C0012 (Double Terminator)<br />
<br />
LacI - EcoR I + Spe I<br />
<br />
DT - EcoR I + Xba I<br />
<br />
Overnight incubation at 37 C followed by 65 C for 15 min (enzyme deactivation)<br />
<br />
Reaction Mixture: <br />
-5 uL template <br />
-5 uL NEB 2 buffer<br />
-0.5 uL BSA<br />
-1 uL restriction enzyme #1<br />
-1 uL restriction enzyme #2<br />
-37.5 uL ddH2O<br />
<br />
===August 6, 2008===<br />
====Nathan Puhl, Roxanne, Alix====<br />
<br />
'''Objective:''' Gel extract digested biobricks to clean for ligation reaction<br />
<br />
Qiagen MinElute Kit was used to gel purify the digested LacI and DT biobricks.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabAugstTeam:University of Lethbridge/Notebook/GeneralLabAugst2008-08-08T01:19:57Z<p>Nathan.puhl: /* Nathan Puhl, Roxanne, Alix */</p>
<hr />
<div>===August 03, 2008===<br />
====Nathan Phillips====<br />
<br />
Tranformation of pUC19:<br />
1. Thaw E.coli DH5a cells on ice<br />
2. Add pUC19 DNA, pipette gently to mix (1μl of plasmid)<br />
3. Let sit for 30 minutes on ice<br />
4. Incubate cells for 30 seconds at 42oC<br />
5. Incubate cells on ice for 2 min<br />
6. Add 1 mL SOC at room temp<br />
7. Incubate for 1 hour at 37oC on shaker<br />
8. Spread 100-300 μl onto a plate made with appropriate antibiotic<br />
9. Grow overnight at 37 °C<br />
<br />
===August 5, 2008===<br />
====Nathan Puhl, Alix, Roxanne and highschool students====<br />
'''Objective:''' Isolate 76bp band from non-specific amplified bands of riboswitch PCR<br />
<br />
Using Qiagen MinElute Kit extracted two very, very similiarly sized bands after running on a 3.5% agarose gel.<br />
<br />
[[Image:purified riboswitch.jpg]]<br />
<br />
'''Objective:''' Cut out LacI gene and open DT vector for construction<br />
<br />
Restriction Digest of BBa_C0012 (LacI) and BBa_C0012 (Double Terminator)<br />
<br />
LacI - EcoR I + Spe I<br />
<br />
DT - EcoR I + Xba I<br />
<br />
Overnight incubation at 37 C followed by 65 C for 15 min (enzyme deactivation)<br />
<br />
Reaction Mixture: <br />
-5 uL template <br />
-5 uL NEB 2 buffer<br />
-0.5 uL BSA<br />
-1 uL restriction enzyme #1<br />
-1 uL restriction enzyme #2<br />
-37.5 uL ddH2O<br />
<br />
===August 6, 2008===<br />
====Nathan Puhl, Roxanne, Alix====<br />
<br />
'''Objective:''' Gel extract digested biobricks to clean for ligation reaction<br />
<br />
Qiagen MinElute Kit was used to gel purify the digested LacI and DT biobricks.</div>Nathan.puhlhttp://2008.igem.org/File:Purified_riboswitch.jpgFile:Purified riboswitch.jpg2008-08-08T01:18:01Z<p>Nathan.puhl: </p>
<hr />
<div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabAugstTeam:University of Lethbridge/Notebook/GeneralLabAugst2008-08-08T01:17:51Z<p>Nathan.puhl: </p>
<hr />
<div>===August 03, 2008===<br />
====Nathan Phillips====<br />
<br />
Tranformation of pUC19:<br />
1. Thaw E.coli DH5a cells on ice<br />
2. Add pUC19 DNA, pipette gently to mix (1μl of plasmid)<br />
3. Let sit for 30 minutes on ice<br />
4. Incubate cells for 30 seconds at 42oC<br />
5. Incubate cells on ice for 2 min<br />
6. Add 1 mL SOC at room temp<br />
7. Incubate for 1 hour at 37oC on shaker<br />
8. Spread 100-300 μl onto a plate made with appropriate antibiotic<br />
9. Grow overnight at 37 °C<br />
<br />
===August 5, 2008===<br />
====Nathan Puhl, Alix, Roxanne and highschool students====<br />
'''Objective:''' Isolate 76bp band from non-specific amplified bands of riboswitch PCR<br />
<br />
Using Qiagen MinElute Kit extracted two very, very similiarly sized bands after running on a 3.5% agarose gel.<br />
<br />
[[Image:purified riboswitch.jpg]]<br />
<br />
'''Objective:''' Cut out LacI gene and open DT vector for construction<br />
<br />
Restriction Digest of BBa_C0012 (LacI) and BBa_C0012 (Double Terminator)<br />
<br />
LacI - EcoR I + Spe I<br />
<br />
DT - EcoR I + Xba I<br />
<br />
Overnight incubation at 37 C followed by 65 C for 15 min (enzyme deactivation)<br />
<br />
Reaction Mixture: <br />
-5 uL template <br />
-5 uL NEB 2 buffer<br />
-0.5 uL BSA<br />
-1 uL restriction enzyme #1<br />
-1 uL restriction enzyme #2<br />
-37.5 uL ddH2O<br />
<br />
===August 6, 2008===<br />
====Nathan Puhl, Roxanne, Alix====<br />
<br />
-Ran a 1% Gel with the Lac I and DT restriction products.<br />
<br />
-Performed a Gel Extraction of the restriction products using the Qiagen Gel Extraction Kit.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJulyTeam:University of Lethbridge/Notebook/GeneralLabJuly2008-07-23T19:13:42Z<p>Nathan.puhl: </p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Phillips, Andrew====<br />
Made 500 mL of LB agar + amp and 500 mL of Liquid LB<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Alix, Sebastian, Munima, Roxanne, Christa====<br />
<br />
Transformed [http://partsregistry.org/wiki/index.php?title=Part:BBa_J24679 BBa_J24679] (RBS + LacI), [http://partsregistry.org/wiki/index.php?title=Part:BBa_P0440 BBa_P0440] (RBS+TetR+T10+T12), leftover DNA from Double T (June 26, 2008), and 1 uL of pSB1A7 plasmid (June 18, 2008).<br />
<br />
Protocol changes: <br />
-3 uL of DNA<br />
-spin down 200 uL of cells and resuspend in 100 uL of LB; plate on LB + amp<br />
<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa, Alix, Roxanne, Sebastian====<br />
Checked transformation plates. Only the positive control (pSB1A7) had colonies (~1500) indicating that there is nothing wrong with the transformation protocol or cells so we must be having problems with the DNA extraction from the filter paper. Next week we will attempt various changes to the protocol to extract more DNA.<br />
<br />
<br />
===July 8, 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
Made 500mL of LB semi-solid media and poured 24 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
====Nathan Puhl, Alix====<br />
Flourescent Reporter<br />
<br />
Transformation from BioBricks LacI (BBA_J24679), TetR (BBa_P0440) and DT (BBa_B0015).<br />
<br />
Protocol:<br />
-punched out 2 spots of filter paper<br />
-15uL TE, for 30 min. @ 50 C<br />
-centrifuge 3 min. @ 15000 g<br />
-freeze 5 min. in -20 C freezer<br />
-heat shock 1 min. in 42 C water bath<br />
-centrifuge 3 min.<br />
-2 uL of plasmid into 25 uL DH5alpha<br />
-leave on ice for 30 min. <br />
-(Left remaining DT, TetR, LacI to sit overnight at room temp. testing for better DNA recovery)<br />
-put in 42 C water bath for 45 sec.<br />
-chill on ice for 2 min.<br />
-add 1 mL of SOC broth<br />
-incubate cells @ 37 C, 225 rpm for 60 min.<br />
-spin down 400 uL cells (1 min. 16000xG), remove 300 uL<br />
-resuspend<br />
-plate, incubate at 37 C overnight<br />
<br />
Subcultured 3 biobricks (for flourescent reporter) glycerol stocked from last year into 5 mL liquid LB + Amp.<br />
-RFP Sub. (BBa_I13507)<br />
-pLACI (BBa_R0011)<br />
-pSTRONG (BBa_J23119)<br />
<br />
loading dye [http://openwetware.org/wiki/Agarose_gel_loading_dye sizes]<br />
<br />
===July 14, 2008===<br />
====Munima, Nathan Puhl, Alix, Andrew====<br />
Ran gel electrophoresis on "comp RP1616 + pSB1A7" cells, LacI, TetR and DT (from July 8/08).<br />
No DNA bands were observed.<br />
<br />
<br />
<br />
===July 15, 2008===<br />
====Nathan Puhl, Andrew, Alix====<br />
Objective: Working on fluorescent reporter system; trying transformation from BioBricks again.<br />
Using: LacI (BBa_J24679) and DT (BBa_B0015)<br />
<br />
Protocol - "Transformation - F00 v.1" (Glinko Bioworks):<br />
<br />
1. Get DNA from filter paper.<br />
2. Place punch directly in 25 uL DH5alpha cells.<br />
3. Leave on ice for 30 mins.<br />
4. 45 second heat shock in 42 C water bath.<br />
5. Chill on ice for 2 minutes.<br />
6. Add 500 uL SOC broth.<br />
7. Incubate at 37 C for 1 hour.<br />
8. Spin down all cells.<br />
9. Remove 400 uL.<br />
10. Resuspend and plate on LB + Amp. Allow to incubate at 37 C overnight.<br />
<br />
<br />
===July 16, 2008===<br />
====Andrew, Alix====<br />
Objective: Assess transformation success <br />
- observed growth on LacI and DT plates<br />
- grew up cells with LB + Amp<br />
- put into shaker incubator at 37 C overnight<br />
<br />
===July 17, 2008===<br />
====Nathan Puhl, Alix, Munima, Christa====<br />
Objective: Assess transformation success and isolate a single colony for subculture<br />
- Streaked colonies onto Brent's LB + Amp plates because there was too much growth on the LacI/DT plates<br />
<br />
====Munima, Christa====<br />
Made 500mL of LB + amp semi-solid media and poured 25 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
<br />
===July 21, 2008===<br />
====Nathan Puhl====<br />
Objective: Subculture transformed cells for plasmid isolation<br />
- Subcultured single colony of re-streaked transformants in LB + amp and shaker incubated overnight at 37 C<br />
<br />
===July 22, 2008===<br />
====Nathan Puhl, Andrew====<br />
Objective:Isolate plasmid from subcultured transformed cells and attempt to transform three more biobricks from the filter paper into ''E.coli''<br />
- Plasmid prepped BBa_J24679 (LacI) and BBa_B0015 (DT) from transformed DH5-alpha<br />
- Transformed DH5-alpha with BBa_P0440 (TetR), BBa_I714062 (GTPTT), and BBa_J31007 (TetA) as per the protocol from July 15, 2008</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJulyTeam:University of Lethbridge/Notebook/GeneralLabJuly2008-07-23T19:12:43Z<p>Nathan.puhl: /* Nathan Puhl, Andrew */</p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Phillips, Andrew====<br />
Made 500 mL of LB agar + amp and 500 mL of Liquid LB<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Alix, Sebastian, Munima, Roxanne, Christa====<br />
<br />
Transformed [http://partsregistry.org/wiki/index.php?title=Part:BBa_J24679 BBa_J24679] (RBS + LacI), [http://partsregistry.org/wiki/index.php?title=Part:BBa_P0440 BBa_P0440] (RBS+TetR+T10+T12), leftover DNA from Double T (June 26, 2008), and 1 uL of pSB1A7 plasmid (June 18, 2008).<br />
<br />
Protocol changes: <br />
-3 uL of DNA<br />
-spin down 200 uL of cells and resuspend in 100 uL of LB; plate on LB + amp<br />
<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa, Alix, Roxanne, Sebastian====<br />
Checked transformation plates. Only the positive control (pSB1A7) had colonies (~1500) indicating that there is nothing wrong with the transformation protocol or cells so we must be having problems with the DNA extraction from the filter paper. Next week we will attempt various changes to the protocol to extract more DNA.<br />
<br />
<br />
===July 8, 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
Made 500mL of LB semi-solid media and poured 24 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
====Nathan Puhl, Alix====<br />
Flourescent Reporter<br />
<br />
Transformation from BioBricks LacI (BBA_J24679), TetR (BBa_P0440) and DT (BBa_B0015).<br />
<br />
Protocol:<br />
-punched out 2 spots of filter paper<br />
-15uL TE, for 30 min. @ 50 C<br />
-centrifuge 3 min. @ 15000 g<br />
-freeze 5 min. in -20 C freezer<br />
-heat shock 1 min. in 42 C water bath<br />
-centrifuge 3 min.<br />
-2 uL of plasmid into 25 uL DH5alpha<br />
-leave on ice for 30 min. <br />
-(Left remaining DT, TetR, LacI to sit overnight at room temp. testing for better DNA recovery)<br />
-put in 42 C water bath for 45 sec.<br />
-chill on ice for 2 min.<br />
-add 1 mL of SOC broth<br />
-incubate cells @ 37 C, 225 rpm for 60 min.<br />
-spin down 400 uL cells (1 min. 16000xG), remove 300 uL<br />
-resuspend<br />
-plate, incubate at 37 C overnight<br />
<br />
Subcultured 3 biobricks (for flourescent reporter) glycerol stocked from last year into 5 mL liquid LB + Amp.<br />
-RFP Sub. (BBa_I13507)<br />
-pLACI (BBa_R0011)<br />
-pSTRONG (BBa_J23119)<br />
<br />
loading dye [http://openwetware.org/wiki/Agarose_gel_loading_dye sizes]<br />
<br />
===July 14, 2008===<br />
====Munima, Nathan Puhl, Alix, Andrew====<br />
Ran gel electrophoresis on "comp RP1616 + pSB1A7" cells, LacI, TetR and DT (from July 8/08).<br />
No DNA bands were observed.<br />
<br />
<br />
<br />
===July 15, 2008===<br />
====Nathan Puhl, Andrew, Alix====<br />
Objective: Working on fluorescent reporter system; trying transformation from BioBricks again.<br />
Using: LacI (BBa_J24679) and DT (BBa_B0015)<br />
<br />
Protocol - "Transformation - F00 v.1" (Glinko Bioworks):<br />
<br />
1. Get DNA from filter paper.<br />
2. Place punch directly in 25 uL DH5alpha cells.<br />
3. Leave on ice for 30 mins.<br />
4. 45 second heat shock in 42 C water bath.<br />
5. Chill on ice for 2 minutes.<br />
6. Add 500 uL SOC broth.<br />
7. Incubate at 37 C for 1 hour.<br />
8. Spin down all cells.<br />
9. Remove 400 uL.<br />
10. Resuspend and plate on LB + Amp. Allow to incubate at 37 C overnight.<br />
<br />
<br />
===July 16, 2008===<br />
====Andrew, Alix====<br />
Objective: Assess transformation success <br />
- observed growth on LacI and DT plates<br />
- grew up cells with LB + Amp<br />
- put into shaker incubator at 37 C overnight<br />
<br />
===July 17, 2008===<br />
====Nathan Puhl, Alix, Munima, Christa====<br />
Objective: Assess transformation success and isolate a single colony for subculture<br />
- streaked colonies onto Brent's LB + Amp plates because there was too much growth on the LacI/DT plates<br />
<br />
====Munima, Christa====<br />
Made 500mL of LB + amp semi-solid media and poured 25 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
<br />
===July 21, 2008===<br />
====Nathan Puhl====<br />
Objective: Subculture transformed cells for plasmid isolation<br />
<br />
Subcultured single colony of re-streaked transformants in LB + amp and shaker incubated overnight at 37 C<br />
<br />
===July 22, 2008===<br />
====Nathan Puhl, Andrew====<br />
Objective:Isolate plasmid from subcultured transformed cells and attempt to transform three more biobricks from the filter paper into ''E.coli''<br />
<br />
Plasmid prepped BBa_J24679 (LacI) and BBa_B0015 (DT) from transformed DH5-alpha<br />
<br />
Transformed DH5-alpha with BBa_P0440 (TetR), BBa_I714062 (GTPTT), and BBa_J31007 (TetA) as per the protocol from July 15, 2008</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJulyTeam:University of Lethbridge/Notebook/GeneralLabJuly2008-07-23T19:10:54Z<p>Nathan.puhl: /* Nathan Puhl */</p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Phillips, Andrew====<br />
Made 500 mL of LB agar + amp and 500 mL of Liquid LB<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Alix, Sebastian, Munima, Roxanne, Christa====<br />
<br />
Transformed [http://partsregistry.org/wiki/index.php?title=Part:BBa_J24679 BBa_J24679] (RBS + LacI), [http://partsregistry.org/wiki/index.php?title=Part:BBa_P0440 BBa_P0440] (RBS+TetR+T10+T12), leftover DNA from Double T (June 26, 2008), and 1 uL of pSB1A7 plasmid (June 18, 2008).<br />
<br />
Protocol changes: <br />
-3 uL of DNA<br />
-spin down 200 uL of cells and resuspend in 100 uL of LB; plate on LB + amp<br />
<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa, Alix, Roxanne, Sebastian====<br />
Checked transformation plates. Only the positive control (pSB1A7) had colonies (~1500) indicating that there is nothing wrong with the transformation protocol or cells so we must be having problems with the DNA extraction from the filter paper. Next week we will attempt various changes to the protocol to extract more DNA.<br />
<br />
<br />
===July 8, 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
Made 500mL of LB semi-solid media and poured 24 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
====Nathan Puhl, Alix====<br />
Flourescent Reporter<br />
<br />
Transformation from BioBricks LacI (BBA_J24679), TetR (BBa_P0440) and DT (BBa_B0015).<br />
<br />
Protocol:<br />
-punched out 2 spots of filter paper<br />
-15uL TE, for 30 min. @ 50 C<br />
-centrifuge 3 min. @ 15000 g<br />
-freeze 5 min. in -20 C freezer<br />
-heat shock 1 min. in 42 C water bath<br />
-centrifuge 3 min.<br />
-2 uL of plasmid into 25 uL DH5alpha<br />
-leave on ice for 30 min. <br />
-(Left remaining DT, TetR, LacI to sit overnight at room temp. testing for better DNA recovery)<br />
-put in 42 C water bath for 45 sec.<br />
-chill on ice for 2 min.<br />
-add 1 mL of SOC broth<br />
-incubate cells @ 37 C, 225 rpm for 60 min.<br />
-spin down 400 uL cells (1 min. 16000xG), remove 300 uL<br />
-resuspend<br />
-plate, incubate at 37 C overnight<br />
<br />
Subcultured 3 biobricks (for flourescent reporter) glycerol stocked from last year into 5 mL liquid LB + Amp.<br />
-RFP Sub. (BBa_I13507)<br />
-pLACI (BBa_R0011)<br />
-pSTRONG (BBa_J23119)<br />
<br />
loading dye [http://openwetware.org/wiki/Agarose_gel_loading_dye sizes]<br />
<br />
===July 14, 2008===<br />
====Munima, Nathan Puhl, Alix, Andrew====<br />
Ran gel electrophoresis on "comp RP1616 + pSB1A7" cells, LacI, TetR and DT (from July 8/08).<br />
No DNA bands were observed.<br />
<br />
<br />
<br />
===July 15, 2008===<br />
====Nathan Puhl, Andrew, Alix====<br />
Objective: Working on fluorescent reporter system; trying transformation from BioBricks again.<br />
Using: LacI (BBa_J24679) and DT (BBa_B0015)<br />
<br />
Protocol - "Transformation - F00 v.1" (Glinko Bioworks):<br />
<br />
1. Get DNA from filter paper.<br />
2. Place punch directly in 25 uL DH5alpha cells.<br />
3. Leave on ice for 30 mins.<br />
4. 45 second heat shock in 42 C water bath.<br />
5. Chill on ice for 2 minutes.<br />
6. Add 500 uL SOC broth.<br />
7. Incubate at 37 C for 1 hour.<br />
8. Spin down all cells.<br />
9. Remove 400 uL.<br />
10. Resuspend and plate on LB + Amp. Allow to incubate at 37 C overnight.<br />
<br />
<br />
===July 16, 2008===<br />
====Andrew, Alix====<br />
Objective: Assess transformation success <br />
- observed growth on LacI and DT plates<br />
- grew up cells with LB + Amp<br />
- put into shaker incubator at 37 C overnight<br />
<br />
===July 17, 2008===<br />
====Nathan Puhl, Alix, Munima, Christa====<br />
Objective: Assess transformation success and isolate a single colony for subculture<br />
- streaked colonies onto Brent's LB + Amp plates because there was too much growth on the LacI/DT plates<br />
<br />
====Munima, Christa====<br />
Made 500mL of LB + amp semi-solid media and poured 25 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
<br />
===July 21, 2008===<br />
====Nathan Puhl====<br />
Objective: Subculture transformed cells for plasmid isolation<br />
<br />
Subcultured single colony of re-streaked transformants in LB + amp and shaker incubated overnight at 37 C<br />
<br />
===July 22, 2008===<br />
====Nathan Puhl, Andrew====<br />
Plasma prepped BBa_J24679 (LacI) and BBa_B0015 (DT) from transformed DH5-alpha<br />
<br />
Transformed DH5-alpha with BBa_P0440 (TetR), BBa_I714062 (GTPTT), and BBa_J31007 (TetA) as per the protocol from July 15, 2008</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJulyTeam:University of Lethbridge/Notebook/GeneralLabJuly2008-07-23T19:10:40Z<p>Nathan.puhl: /* Nathan Puhl */</p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Phillips, Andrew====<br />
Made 500 mL of LB agar + amp and 500 mL of Liquid LB<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Alix, Sebastian, Munima, Roxanne, Christa====<br />
<br />
Transformed [http://partsregistry.org/wiki/index.php?title=Part:BBa_J24679 BBa_J24679] (RBS + LacI), [http://partsregistry.org/wiki/index.php?title=Part:BBa_P0440 BBa_P0440] (RBS+TetR+T10+T12), leftover DNA from Double T (June 26, 2008), and 1 uL of pSB1A7 plasmid (June 18, 2008).<br />
<br />
Protocol changes: <br />
-3 uL of DNA<br />
-spin down 200 uL of cells and resuspend in 100 uL of LB; plate on LB + amp<br />
<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa, Alix, Roxanne, Sebastian====<br />
Checked transformation plates. Only the positive control (pSB1A7) had colonies (~1500) indicating that there is nothing wrong with the transformation protocol or cells so we must be having problems with the DNA extraction from the filter paper. Next week we will attempt various changes to the protocol to extract more DNA.<br />
<br />
<br />
===July 8, 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
Made 500mL of LB semi-solid media and poured 24 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
====Nathan Puhl, Alix====<br />
Flourescent Reporter<br />
<br />
Transformation from BioBricks LacI (BBA_J24679), TetR (BBa_P0440) and DT (BBa_B0015).<br />
<br />
Protocol:<br />
-punched out 2 spots of filter paper<br />
-15uL TE, for 30 min. @ 50 C<br />
-centrifuge 3 min. @ 15000 g<br />
-freeze 5 min. in -20 C freezer<br />
-heat shock 1 min. in 42 C water bath<br />
-centrifuge 3 min.<br />
-2 uL of plasmid into 25 uL DH5alpha<br />
-leave on ice for 30 min. <br />
-(Left remaining DT, TetR, LacI to sit overnight at room temp. testing for better DNA recovery)<br />
-put in 42 C water bath for 45 sec.<br />
-chill on ice for 2 min.<br />
-add 1 mL of SOC broth<br />
-incubate cells @ 37 C, 225 rpm for 60 min.<br />
-spin down 400 uL cells (1 min. 16000xG), remove 300 uL<br />
-resuspend<br />
-plate, incubate at 37 C overnight<br />
<br />
Subcultured 3 biobricks (for flourescent reporter) glycerol stocked from last year into 5 mL liquid LB + Amp.<br />
-RFP Sub. (BBa_I13507)<br />
-pLACI (BBa_R0011)<br />
-pSTRONG (BBa_J23119)<br />
<br />
loading dye [http://openwetware.org/wiki/Agarose_gel_loading_dye sizes]<br />
<br />
===July 14, 2008===<br />
====Munima, Nathan Puhl, Alix, Andrew====<br />
Ran gel electrophoresis on "comp RP1616 + pSB1A7" cells, LacI, TetR and DT (from July 8/08).<br />
No DNA bands were observed.<br />
<br />
<br />
<br />
===July 15, 2008===<br />
====Nathan Puhl, Andrew, Alix====<br />
Objective: Working on fluorescent reporter system; trying transformation from BioBricks again.<br />
Using: LacI (BBa_J24679) and DT (BBa_B0015)<br />
<br />
Protocol - "Transformation - F00 v.1" (Glinko Bioworks):<br />
<br />
1. Get DNA from filter paper.<br />
2. Place punch directly in 25 uL DH5alpha cells.<br />
3. Leave on ice for 30 mins.<br />
4. 45 second heat shock in 42 C water bath.<br />
5. Chill on ice for 2 minutes.<br />
6. Add 500 uL SOC broth.<br />
7. Incubate at 37 C for 1 hour.<br />
8. Spin down all cells.<br />
9. Remove 400 uL.<br />
10. Resuspend and plate on LB + Amp. Allow to incubate at 37 C overnight.<br />
<br />
<br />
===July 16, 2008===<br />
====Andrew, Alix====<br />
Objective: Assess transformation success <br />
- observed growth on LacI and DT plates<br />
- grew up cells with LB + Amp<br />
- put into shaker incubator at 37 C overnight<br />
<br />
===July 17, 2008===<br />
====Nathan Puhl, Alix, Munima, Christa====<br />
Objective: Assess transformation success and isolate a single colony for subculture<br />
- streaked colonies onto Brent's LB + Amp plates because there was too much growth on the LacI/DT plates<br />
<br />
====Munima, Christa====<br />
Made 500mL of LB + amp semi-solid media and poured 25 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
<br />
===July 21, 2008===<br />
====Nathan Puhl====<br />
Objective: Subculture transformamed cells for plasmid isolation<br />
Subcultured single colony of re-streaked transformants in LB + amp and shaker incubated overnight at 37 C<br />
<br />
===July 22, 2008===<br />
====Nathan Puhl, Andrew====<br />
Plasma prepped BBa_J24679 (LacI) and BBa_B0015 (DT) from transformed DH5-alpha<br />
<br />
Transformed DH5-alpha with BBa_P0440 (TetR), BBa_I714062 (GTPTT), and BBa_J31007 (TetA) as per the protocol from July 15, 2008</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJulyTeam:University of Lethbridge/Notebook/GeneralLabJuly2008-07-23T19:10:03Z<p>Nathan.puhl: /* Nathan Puhl, Alix, Munima, Christa */</p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Phillips, Andrew====<br />
Made 500 mL of LB agar + amp and 500 mL of Liquid LB<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Alix, Sebastian, Munima, Roxanne, Christa====<br />
<br />
Transformed [http://partsregistry.org/wiki/index.php?title=Part:BBa_J24679 BBa_J24679] (RBS + LacI), [http://partsregistry.org/wiki/index.php?title=Part:BBa_P0440 BBa_P0440] (RBS+TetR+T10+T12), leftover DNA from Double T (June 26, 2008), and 1 uL of pSB1A7 plasmid (June 18, 2008).<br />
<br />
Protocol changes: <br />
-3 uL of DNA<br />
-spin down 200 uL of cells and resuspend in 100 uL of LB; plate on LB + amp<br />
<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa, Alix, Roxanne, Sebastian====<br />
Checked transformation plates. Only the positive control (pSB1A7) had colonies (~1500) indicating that there is nothing wrong with the transformation protocol or cells so we must be having problems with the DNA extraction from the filter paper. Next week we will attempt various changes to the protocol to extract more DNA.<br />
<br />
<br />
===July 8, 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
Made 500mL of LB semi-solid media and poured 24 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
====Nathan Puhl, Alix====<br />
Flourescent Reporter<br />
<br />
Transformation from BioBricks LacI (BBA_J24679), TetR (BBa_P0440) and DT (BBa_B0015).<br />
<br />
Protocol:<br />
-punched out 2 spots of filter paper<br />
-15uL TE, for 30 min. @ 50 C<br />
-centrifuge 3 min. @ 15000 g<br />
-freeze 5 min. in -20 C freezer<br />
-heat shock 1 min. in 42 C water bath<br />
-centrifuge 3 min.<br />
-2 uL of plasmid into 25 uL DH5alpha<br />
-leave on ice for 30 min. <br />
-(Left remaining DT, TetR, LacI to sit overnight at room temp. testing for better DNA recovery)<br />
-put in 42 C water bath for 45 sec.<br />
-chill on ice for 2 min.<br />
-add 1 mL of SOC broth<br />
-incubate cells @ 37 C, 225 rpm for 60 min.<br />
-spin down 400 uL cells (1 min. 16000xG), remove 300 uL<br />
-resuspend<br />
-plate, incubate at 37 C overnight<br />
<br />
Subcultured 3 biobricks (for flourescent reporter) glycerol stocked from last year into 5 mL liquid LB + Amp.<br />
-RFP Sub. (BBa_I13507)<br />
-pLACI (BBa_R0011)<br />
-pSTRONG (BBa_J23119)<br />
<br />
loading dye [http://openwetware.org/wiki/Agarose_gel_loading_dye sizes]<br />
<br />
===July 14, 2008===<br />
====Munima, Nathan Puhl, Alix, Andrew====<br />
Ran gel electrophoresis on "comp RP1616 + pSB1A7" cells, LacI, TetR and DT (from July 8/08).<br />
No DNA bands were observed.<br />
<br />
<br />
<br />
===July 15, 2008===<br />
====Nathan Puhl, Andrew, Alix====<br />
Objective: Working on fluorescent reporter system; trying transformation from BioBricks again.<br />
Using: LacI (BBa_J24679) and DT (BBa_B0015)<br />
<br />
Protocol - "Transformation - F00 v.1" (Glinko Bioworks):<br />
<br />
1. Get DNA from filter paper.<br />
2. Place punch directly in 25 uL DH5alpha cells.<br />
3. Leave on ice for 30 mins.<br />
4. 45 second heat shock in 42 C water bath.<br />
5. Chill on ice for 2 minutes.<br />
6. Add 500 uL SOC broth.<br />
7. Incubate at 37 C for 1 hour.<br />
8. Spin down all cells.<br />
9. Remove 400 uL.<br />
10. Resuspend and plate on LB + Amp. Allow to incubate at 37 C overnight.<br />
<br />
<br />
===July 16, 2008===<br />
====Andrew, Alix====<br />
Objective: Assess transformation success <br />
- observed growth on LacI and DT plates<br />
- grew up cells with LB + Amp<br />
- put into shaker incubator at 37 C overnight<br />
<br />
===July 17, 2008===<br />
====Nathan Puhl, Alix, Munima, Christa====<br />
Objective: Assess transformation success and isolate a single colony for subculture<br />
- streaked colonies onto Brent's LB + Amp plates because there was too much growth on the LacI/DT plates<br />
<br />
====Munima, Christa====<br />
Made 500mL of LB + amp semi-solid media and poured 25 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
<br />
===July 21, 2008===<br />
====Nathan Puhl====<br />
Subcultured single colony of re-streaked transformants in LB + amp and shaker incubated overnight at 37 C<br />
<br />
<br />
===July 22, 2008===<br />
====Nathan Puhl, Andrew====<br />
Plasma prepped BBa_J24679 (LacI) and BBa_B0015 (DT) from transformed DH5-alpha<br />
<br />
Transformed DH5-alpha with BBa_P0440 (TetR), BBa_I714062 (GTPTT), and BBa_J31007 (TetA) as per the protocol from July 15, 2008</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJulyTeam:University of Lethbridge/Notebook/GeneralLabJuly2008-07-23T19:09:19Z<p>Nathan.puhl: /* Andrew, Alix */</p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Phillips, Andrew====<br />
Made 500 mL of LB agar + amp and 500 mL of Liquid LB<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Alix, Sebastian, Munima, Roxanne, Christa====<br />
<br />
Transformed [http://partsregistry.org/wiki/index.php?title=Part:BBa_J24679 BBa_J24679] (RBS + LacI), [http://partsregistry.org/wiki/index.php?title=Part:BBa_P0440 BBa_P0440] (RBS+TetR+T10+T12), leftover DNA from Double T (June 26, 2008), and 1 uL of pSB1A7 plasmid (June 18, 2008).<br />
<br />
Protocol changes: <br />
-3 uL of DNA<br />
-spin down 200 uL of cells and resuspend in 100 uL of LB; plate on LB + amp<br />
<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa, Alix, Roxanne, Sebastian====<br />
Checked transformation plates. Only the positive control (pSB1A7) had colonies (~1500) indicating that there is nothing wrong with the transformation protocol or cells so we must be having problems with the DNA extraction from the filter paper. Next week we will attempt various changes to the protocol to extract more DNA.<br />
<br />
<br />
===July 8, 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
Made 500mL of LB semi-solid media and poured 24 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
====Nathan Puhl, Alix====<br />
Flourescent Reporter<br />
<br />
Transformation from BioBricks LacI (BBA_J24679), TetR (BBa_P0440) and DT (BBa_B0015).<br />
<br />
Protocol:<br />
-punched out 2 spots of filter paper<br />
-15uL TE, for 30 min. @ 50 C<br />
-centrifuge 3 min. @ 15000 g<br />
-freeze 5 min. in -20 C freezer<br />
-heat shock 1 min. in 42 C water bath<br />
-centrifuge 3 min.<br />
-2 uL of plasmid into 25 uL DH5alpha<br />
-leave on ice for 30 min. <br />
-(Left remaining DT, TetR, LacI to sit overnight at room temp. testing for better DNA recovery)<br />
-put in 42 C water bath for 45 sec.<br />
-chill on ice for 2 min.<br />
-add 1 mL of SOC broth<br />
-incubate cells @ 37 C, 225 rpm for 60 min.<br />
-spin down 400 uL cells (1 min. 16000xG), remove 300 uL<br />
-resuspend<br />
-plate, incubate at 37 C overnight<br />
<br />
Subcultured 3 biobricks (for flourescent reporter) glycerol stocked from last year into 5 mL liquid LB + Amp.<br />
-RFP Sub. (BBa_I13507)<br />
-pLACI (BBa_R0011)<br />
-pSTRONG (BBa_J23119)<br />
<br />
loading dye [http://openwetware.org/wiki/Agarose_gel_loading_dye sizes]<br />
<br />
===July 14, 2008===<br />
====Munima, Nathan Puhl, Alix, Andrew====<br />
Ran gel electrophoresis on "comp RP1616 + pSB1A7" cells, LacI, TetR and DT (from July 8/08).<br />
No DNA bands were observed.<br />
<br />
<br />
<br />
===July 15, 2008===<br />
====Nathan Puhl, Andrew, Alix====<br />
Objective: Working on fluorescent reporter system; trying transformation from BioBricks again.<br />
Using: LacI (BBa_J24679) and DT (BBa_B0015)<br />
<br />
Protocol - "Transformation - F00 v.1" (Glinko Bioworks):<br />
<br />
1. Get DNA from filter paper.<br />
2. Place punch directly in 25 uL DH5alpha cells.<br />
3. Leave on ice for 30 mins.<br />
4. 45 second heat shock in 42 C water bath.<br />
5. Chill on ice for 2 minutes.<br />
6. Add 500 uL SOC broth.<br />
7. Incubate at 37 C for 1 hour.<br />
8. Spin down all cells.<br />
9. Remove 400 uL.<br />
10. Resuspend and plate on LB + Amp. Allow to incubate at 37 C overnight.<br />
<br />
<br />
===July 16, 2008===<br />
====Andrew, Alix====<br />
Objective: Assess transformation success <br />
- observed growth on LacI and DT plates<br />
- grew up cells with LB + Amp<br />
- put into shaker incubator at 37 C overnight<br />
<br />
===July 17, 2008===<br />
====Nathan Puhl, Alix, Munima, Christa====<br />
- streaked colonies onto Brent's LB + Amp plates because there was too much growth on the LacI/DT plates<br />
<br />
====Munima, Christa====<br />
Made 500mL of LB + amp semi-solid media and poured 25 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
<br />
===July 21, 2008===<br />
====Nathan Puhl====<br />
Subcultured single colony of re-streaked transformants in LB + amp and shaker incubated overnight at 37 C<br />
<br />
<br />
===July 22, 2008===<br />
====Nathan Puhl, Andrew====<br />
Plasma prepped BBa_J24679 (LacI) and BBa_B0015 (DT) from transformed DH5-alpha<br />
<br />
Transformed DH5-alpha with BBa_P0440 (TetR), BBa_I714062 (GTPTT), and BBa_J31007 (TetA) as per the protocol from July 15, 2008</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/ProjectTeam:University of Lethbridge/Project2008-07-23T19:07:18Z<p>Nathan.puhl: </p>
<hr />
<div>__NOTOC__<br />
<br />
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<span style="background-color:#000000; color:white"><!--__TOC__--></span><br />
<br />
==<span style="background-color:#000000; color:white"> '''The “Bacuum" Cleaner – an intelligent self-propelling keener cleaner'''</span> ==<br />
<br />
<!--- The Mission, Experiments ---><br />
Our goal is to create a modified ''Escherichia coli'' bacterium capable of seeking out and degrading toxic aromatic pollutants created during the oil refinery and mining processes. Our “Bacuum" cleaner will respond to this destructive compound through interaction with a programmed riboswitch (an mRNA capable of binding a target molecule with its 5’ UTR which causes a conformational change, thereby regulating any downstream protein translation). By using riboswitches that switch at varying concentrations of target ligand, we can alter the signal which is induced. At low concentrations, we intend to have our riboswitch express the motility protein cheZ in ''E. coli'', thus directing the bacterium towards higher concentrations of our target molecule (i.e. positive chemotaxis). Once it reaches a threshold concentration, a catabolic pathway capable of degrading our target pollutant will be activated.<br />
<br />
We plan to use [http://en.wikipedia.org/wiki/Systematic_Evolution_of_Ligands_by_Exponential_Enrichment <font color=yellow>SELEX</font>] to reprogram the theophylline riboswitch initially characterized by our team last year, aiming to have it bind one of many toxic aromatic compounds. This not only builds on our past work with riboswitches but also demonstrates an alternative function for riboswitch-controlled chemotaxis, changing from a simple detection to active “search and destroy” role. Fluorescent protein expression will be used to demonstrate and characterize the functionality of the riboswitch.<br />
<br />
Our project also has major environmental implications, especially in Alberta, where the oil industry is the driving economic sector. The tailings ponds used to house discarded mining refuse from the oil refineries pose a major environmental dilemma. The problem arises with being unable to collect and degrade the compounds which make up these toxic soups. These not only toxic but often corrosive water beds not only affect the local environment but can also cause severe repercussions to other ecosystems as much of the wildlife relies on getting their food and water from the now disrupted bionetwork. These tailings ponds are often laced with heavy metals and dangerous chemicals, including these toxic aromatic hydrocarbons which can be extremely difficult to breakdown. Despite the efforts of the corporations responsible, some of these chemicals can leech into and contaminate the surrounding ground water and soil. It is our intention that our “Bacuum” will not only remove these compounds from such an aqueous system, but will also eliminate them. This goes one step further than the ordinary less-intelligent bag-dependent vacuum cleaners. Ultimately we aim to have our bacteria convert the target aromatic into a useable compound. One example would be the biosynthesis of fatty acids as an alternative fuel source, creating a potential “Bioreactor” which is capable of creating a more environmentally friendly fuel source.<br />
<br />
== <span style="background-color:#000000; color:white">Project Details</span>==<br />
<br />
<br />
<br />
<br />
<br />
=== <span style="background-color:#000000; color:white">Chemotaxis - the "search" from "search and destroy" </span>===<br />
<br />
Our subproject is to ensure our 'bacuum cleaner' will search out and move towards our potential target. Using a riboswitch which responds to this target, a gene involved in motility in ''Escherichia coli'' (''CheZ'') will be switched to an 'on' state. ''CheZ'' controls bacterial movement by dephosphorylating ''CheY'', altering the bacteria from random tumbling to directed movement. By doing so, we hope to observe the bacteria moving towards the target molecule in a positive chemotaxis manner. <br />
<br />
Because we wish to eventually have our bacteria degrade this activating compound, we need to have a multi-level response system established. Our bacteria should respond to low levels of the target by activating our ''CheZ'' riboswitch (through a strong binding aptamer) and to high levels by activating the catabolic pathway responsible for its degradation. By doing so, we will have created our 'search and destroy' system.<br />
<br />
Our subgroup will attempt to establish a working motility assay to prove control of chemotaxis is possible with an already characterized theophylline-binding riboswitch and later with our new target molecule. We will also use a colour read-out system of two fluorescent proteins to demonstrate the differential binding of the ligand at varying concentrations.<br />
<br />
=== <span style="background-color:#000000; color:white">Riboswitch Characterization</span>===<br />
<br />
This subproject was created to quantify and characterize the binding affinity of the theophylline riboswitch to theophylline as well as other similar aromatic ligands. We will use Green Fluorescent Protein (GFP) sequenced behind the riboswitch to help in this endeavour. When the target ligand is bound to the riboswitch, the production of GFP will be induced, producing green colonies. When the ligand is not bound, GFP will not be produced. We intend to try to characterize the riboswitch with and without the iGEM scar to determine if this produces any effect on the relative effeciency of the riboswitch on the protein.<br />
<br />
We will use variable concentrations of Theophylline, Caffeine and 3-methylxanthine to help with the characterization of the initial theophylline riboswitch. We will also test other aromatic compounds to determine wether or not, the theophylline riboswitch can be utilized to bind them as well. <br />
<br />
Once we have successfully produced mutant riboswitches through SELEX, will we then perform these same characterization experiments to determine their binding affinities.<br />
<br />
[[Image:riboswitch.jpeg|650px|center]]<br />
<br />
===<span style="background-color:#000000; color:white"> SELEX </span>===<br />
<br />
Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a method used to produce novel nucleic acid sequences, referred to as aptamers, that bind specific ligands. SELEX involves screening a library of possible aptamers for binding capability repeatedly until a sequence of the desired specificity or binding affinity is found.<br />
<br />
SELEX will be used in our project to produce multiple riboswitches capable of specifically binding the target toxin at varying concentrations. Multiple riboswitches are needed to allow a separation between the "search" and the "destroy" functions of our Bacuum cleaner, with the CheZ search ability controlled at low ligand levels and the degradation pathway controlled at high levels.<br />
<br />
=== <span style="background-color:#000000; color:white">TIR </span>===<br />
<br />
Translation initiation regions (TIRs) are elements found in certain eukaryotic mRNAs that facilitate translation initiation by noncanonical, end independent interactions. We are going to incorporate the TIR from the rpsA gene, encoding ribosomal protein S1, of E. coli into our Bacuum cleaner with green fluoescent protein as a means of monitoring the location of our cells using minimal cell resources. <br />
<br />
The TIR from the rpsA gene will be characterized through the comparison of translation rates for GFP to other canonical RBS. This will allow us to standardize the number of ribosomal initiations per second (RiPS). In generating these parameters we will be able to contribute to building a database of standard parts that can be used to generate biological systems that have a greater degree of reliablity.<br />
<br />
== <span style="background-color:#000000; color:white">Results </span>==<br />
<br />
<br />
----<br />
<br />
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<br />
<br />
<br />
</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJulyTeam:University of Lethbridge/Notebook/GeneralLabJuly2008-07-23T01:55:53Z<p>Nathan.puhl: </p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Phillips, Andrew====<br />
Made 500 mL of LB agar + amp and 500 mL of Liquid LB<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Alix, Sebastian, Munima, Roxanne, Christa====<br />
<br />
Transformed [http://partsregistry.org/wiki/index.php?title=Part:BBa_J24679 BBa_J24679] (RBS + LacI), [http://partsregistry.org/wiki/index.php?title=Part:BBa_P0440 BBa_P0440] (RBS+TetR+T10+T12), leftover DNA from Double T (June 26, 2008), and 1 uL of pSB1A7 plasmid (June 18, 2008).<br />
<br />
Protocol changes: <br />
-3 uL of DNA<br />
-spin down 200 uL of cells and resuspend in 100 uL of LB; plate on LB + amp<br />
<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa, Alix, Roxanne, Sebastian====<br />
Checked transformation plates. Only the positive control (pSB1A7) had colonies (~1500) indicating that there is nothing wrong with the transformation protocol or cells so we must be having problems with the DNA extraction from the filter paper. Next week we will attempt various changes to the protocol to extract more DNA.<br />
<br />
<br />
===July 8, 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
Made 500mL of LB semi-solid media and poured 24 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
====Nathan Puhl, Alix====<br />
Flourescent Reporter<br />
<br />
Transformation from BioBricks LacI (BBA_J24679), TetR (BBa_P0440) and DT (BBa_B0015).<br />
<br />
Protocol:<br />
-punched out 2 spots of filter paper<br />
-15uL TE, for 30 min. @ 50 C<br />
-centrifuge 3 min. @ 15000 g<br />
-freeze 5 min. in -20 C freezer<br />
-heat shock 1 min. in 42 C water bath<br />
-centrifuge 3 min.<br />
-2 uL of plasmid into 25 uL DH5alpha<br />
-leave on ice for 30 min. <br />
-(Left remaining DT, TetR, LacI to sit overnight at room temp. testing for better DNA recovery)<br />
-put in 42 C water bath for 45 sec.<br />
-chill on ice for 2 min.<br />
-add 1 mL of SOC broth<br />
-incubate cells @ 37 C, 225 rpm for 60 min.<br />
-spin down 400 uL cells (1 min. 16000xG), remove 300 uL<br />
-resuspend<br />
-plate, incubate at 37 C overnight<br />
<br />
Subcultured 3 biobricks (for flourescent reporter) glycerol stocked from last year into 5 mL liquid LB + Amp.<br />
-RFP Sub. (BBa_I13507)<br />
-pLACI (BBa_R0011)<br />
-pSTRONG (BBa_J23119)<br />
<br />
loading dye [http://openwetware.org/wiki/Agarose_gel_loading_dye sizes]<br />
<br />
===July 14, 2008===<br />
====Munima, Nathan Puhl, Alix, Andrew====<br />
Ran gel electrophoresis on "comp RP1616 + pSB1A7" cells, LacI, TetR and DT (from July 8/08).<br />
No DNA bands were observed.<br />
<br />
<br />
<br />
===July 15, 2008===<br />
====Nathan Puhl, Andrew, Alix====<br />
Objective: Working on fluorescent reporter system; trying transformation from BioBricks again.<br />
Using: LacI (BBa_J24679) and DT (BBa_B0015)<br />
<br />
Protocol - "Transformation - F00 v.1" (Glinko Bioworks):<br />
<br />
1. Get DNA from filter paper.<br />
2. Place punch directly in 25 uL DH5alpha cells.<br />
3. Leave on ice for 30 mins.<br />
4. 45 second heat shock in 42 C water bath.<br />
5. Chill on ice for 2 minutes.<br />
6. Add 500 uL SOC broth.<br />
7. Incubate at 37 C for 1 hour.<br />
8. Spin down all cells.<br />
9. Remove 400 uL.<br />
10. Resuspend and plate on LB + Amp. Allow to incubate at 37 C overnight.<br />
<br />
<br />
===July 16, 2008===<br />
====Andrew, Alix====<br />
- observed growth on LacI and DT plates<br />
- grew up cells with LB + Amp<br />
- put into shaker incubator at 37 C overnight<br />
<br />
<br />
===July 17, 2008===<br />
====Nathan Puhl, Alix, Munima, Christa====<br />
- streaked colonies onto Brent's LB + Amp plates because there was too much growth on the LacI/DT plates<br />
<br />
====Munima, Christa====<br />
Made 500mL of LB + amp semi-solid media and poured 25 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
<br />
===July 21, 2008===<br />
====Nathan Puhl====<br />
Subcultured single colony of re-streaked transformants in LB + amp and shaker incubated overnight at 37 C<br />
<br />
<br />
===July 22, 2008===<br />
====Nathan Puhl, Andrew====<br />
Plasma prepped BBa_J24679 (LacI) and BBa_B0015 (DT) from transformed DH5-alpha<br />
<br />
Transformed DH5-alpha with BBa_P0440 (TetR), BBa_I714062 (GTPTT), and BBa_J31007 (TetA) as per the protocol from July 15, 2008</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJulyTeam:University of Lethbridge/Notebook/GeneralLabJuly2008-07-23T01:52:50Z<p>Nathan.puhl: /* Nathan Puhl, Alix */</p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Phillips, Andrew====<br />
Made 500 mL of LB agar + amp and 500 mL of Liquid LB<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Alix, Sebastian, Munima, Roxanne, Christa====<br />
<br />
Transformed [http://partsregistry.org/wiki/index.php?title=Part:BBa_J24679 BBa_J24679] (RBS + LacI), [http://partsregistry.org/wiki/index.php?title=Part:BBa_P0440 BBa_P0440] (RBS+TetR+T10+T12), leftover DNA from Double T (June 26, 2008), and 1 uL of pSB1A7 plasmid (June 18, 2008).<br />
<br />
Protocol changes: <br />
-3 uL of DNA<br />
-spin down 200 uL of cells and resuspend in 100 uL of LB; plate on LB + amp<br />
<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa, Alix, Roxanne, Sebastian====<br />
Checked transformation plates. Only the positive control (pSB1A7) had colonies (~1500) indicating that there is nothing wrong with the transformation protocol or cells so we must be having problems with the DNA extraction from the filter paper. Next week we will attempt various changes to the protocol to extract more DNA.<br />
<br />
<br />
===July 8, 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
Made 500mL of LB semi-solid media and poured 24 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
====Nathan Puhl, Alix====<br />
Flourescent Reporter<br />
<br />
Transformation from BioBricks LacI (BBA_J24679), TetR (BBa_P0440) and DT (BBa_B0015).<br />
<br />
Protocol:<br />
-punched out 2 spots of filter paper<br />
-15uL TE, for 30 min. @ 50 C<br />
-centrifuge 3 min. @ 15000 g<br />
-freeze 5 min. in -20 C freezer<br />
-heat shock 1 min. in 42 C water bath<br />
-centrifuge 3 min.<br />
-2 uL of plasmid into 25 uL DH5alpha<br />
-leave on ice for 30 min. <br />
-(Left remaining DT, TetR, LacI to sit overnight at room temp. testing for better DNA recovery)<br />
-put in 42 C water bath for 45 sec.<br />
-chill on ice for 2 min.<br />
-add 1 mL of SOC broth<br />
-incubate cells @ 37 C, 225 rpm for 60 min.<br />
-spin down 400 uL cells (1 min. 16000xG), remove 300 uL<br />
-resuspend<br />
-plate, incubate at 37 C overnight<br />
<br />
Subcultured 3 biobricks (for flourescent reporter) glycerol stocked from last year into 5 mL liquid LB + Amp.<br />
-RFP Sub. (BBa_I13507)<br />
-pLACI (BBa_R0011)<br />
-pSTRONG (BBa_J23119)<br />
<br />
loading dye [http://openwetware.org/wiki/Agarose_gel_loading_dye sizes]<br />
<br />
===July 14, 2008===<br />
====Munima, Nathan Puhl, Alix, Andrew====<br />
Ran gel electrophoresis on "comp RP1616 + pSB1A7" cells, LacI, TetR and DT (from July 8/08).<br />
No DNA bands were observed.<br />
<br />
<br />
<br />
===July 15, 2008===<br />
====Nathan Puhl, Andrew, Alix====<br />
Objective: Working on fluorescent reporter system; trying transformation from BioBricks again.<br />
Using: LacI (BBa_J24679) and DT (BBa_B0015)<br />
<br />
Protocol - "Transformation - F00 v.1" (Glinko Bioworks):<br />
<br />
1. Get DNA from filter paper.<br />
2. Place punch directly in 25 uL DH5alpha cells.<br />
3. Leave on ice for 30 mins.<br />
4. 45 second heat shock in 42 C water bath.<br />
5. Chill on ice for 2 minutes.<br />
6. Add 500 uL SOC broth.<br />
7. Incubate at 37 C for 1 hour.<br />
8. Spin down all cells.<br />
9. Remove 400 uL.<br />
10. Resuspend and plate on LB + Amp. Allow to incubate at 37 C overnight.<br />
<br />
<br />
===July 16, 2008===<br />
====Andrew, Alix====<br />
- observed growth on LacI and DT plates<br />
- grew up cells with LB + Amp<br />
- put into shaker incubator at 37 C overnight<br />
<br />
<br />
===July 17, 2008===<br />
====Nathan Puhl, Alix, Munima, Christa====<br />
- streaked colonies onto Brent's LB + Amp plates because there was too much growth on the LacI/DT plates<br />
<br />
====Munima, Christa====<br />
Made 500mL of LB + amp semi-solid media and poured 25 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
<br />
===July 21, 2008===<br />
====Nathan Puhl====<br />
Subcultured single colony of re-streaked transformants in LB + amp and shaker incubated overnight at 37 C</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJulyTeam:University of Lethbridge/Notebook/GeneralLabJuly2008-07-23T01:52:01Z<p>Nathan.puhl: /* Nathan Puhl, Alix */</p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Phillips, Andrew====<br />
Made 500 mL of LB agar + amp and 500 mL of Liquid LB<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Alix, Sebastian, Munima, Roxanne, Christa====<br />
<br />
Transformed [http://partsregistry.org/wiki/index.php?title=Part:BBa_J24679 BBa_J24679] (RBS + LacI), [http://partsregistry.org/wiki/index.php?title=Part:BBa_P0440 BBa_P0440] (RBS+TetR+T10+T12), leftover DNA from Double T (June 26, 2008), and 1 uL of pSB1A7 plasmid (June 18, 2008).<br />
<br />
Protocol changes: <br />
-3 uL of DNA<br />
-spin down 200 uL of cells and resuspend in 100 uL of LB; plate on LB + amp<br />
<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa, Alix, Roxanne, Sebastian====<br />
Checked transformation plates. Only the positive control (pSB1A7) had colonies (~1500) indicating that there is nothing wrong with the transformation protocol or cells so we must be having problems with the DNA extraction from the filter paper. Next week we will attempt various changes to the protocol to extract more DNA.<br />
<br />
<br />
===July 8, 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
Made 500mL of LB semi-solid media and poured 24 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
====Nathan Puhl, Alix====<br />
Flourescent Reporter<br />
<br />
Transformation from BioBricks LacI (BBA_J24679), TetR (BBa_P0440) and DT (BBa_B0015).<br />
<br />
Protocol:<br />
-punched out 2 spots of filter paper<br />
-15uL TE, for 30 min. @ 50 C<br />
-centrifuge 3 min. @ 15000 g<br />
-freeze 5 min. in -20 C freezer<br />
-heat shock 1 min. in 42 C water bath<br />
-centrifuge 3 min.<br />
-2 uL of plasmid into 25 uL DH5alpha<br />
-leave on ice for 30 min. (Left remaining DT, TetR, LacI to sit overnight at room temp. to possibly test for <br />
<br />
better DNA recovery)<br />
-put in 42 C water bath for 45 sec.<br />
-chill on ice for 2 min.<br />
-add 1 mL of SOC broth<br />
-incubate cells @ 37 C, 225 rpm for 60 min.<br />
-spin down 400 uL cells (1 min. 16000xG), remove 300 uL<br />
-resuspend<br />
-plate, incubate at 37 C overnight<br />
<br />
Subcultured 3 biobricks (for flourescent reporter) glycerol stocked from last year into 5 mL liquid LB + Amp.<br />
-RFP Sub. (BBa_I13507)<br />
-pLACI (BBa_R0011)<br />
-pSTRONG (BBa_J23119)<br />
<br />
loading dye [http://openwetware.org/wiki/Agarose_gel_loading_dye sizes]<br />
<br />
===July 14, 2008===<br />
====Munima, Nathan Puhl, Alix, Andrew====<br />
Ran gel electrophoresis on "comp RP1616 + pSB1A7" cells, LacI, TetR and DT (from July 8/08).<br />
No DNA bands were observed.<br />
<br />
<br />
<br />
===July 15, 2008===<br />
====Nathan Puhl, Andrew, Alix====<br />
Objective: Working on fluorescent reporter system; trying transformation from BioBricks again.<br />
Using: LacI (BBa_J24679) and DT (BBa_B0015)<br />
<br />
Protocol - "Transformation - F00 v.1" (Glinko Bioworks):<br />
<br />
1. Get DNA from filter paper.<br />
2. Place punch directly in 25 uL DH5alpha cells.<br />
3. Leave on ice for 30 mins.<br />
4. 45 second heat shock in 42 C water bath.<br />
5. Chill on ice for 2 minutes.<br />
6. Add 500 uL SOC broth.<br />
7. Incubate at 37 C for 1 hour.<br />
8. Spin down all cells.<br />
9. Remove 400 uL.<br />
10. Resuspend and plate on LB + Amp. Allow to incubate at 37 C overnight.<br />
<br />
<br />
===July 16, 2008===<br />
====Andrew, Alix====<br />
- observed growth on LacI and DT plates<br />
- grew up cells with LB + Amp<br />
- put into shaker incubator at 37 C overnight<br />
<br />
<br />
===July 17, 2008===<br />
====Nathan Puhl, Alix, Munima, Christa====<br />
- streaked colonies onto Brent's LB + Amp plates because there was too much growth on the LacI/DT plates<br />
<br />
====Munima, Christa====<br />
Made 500mL of LB + amp semi-solid media and poured 25 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
<br />
===July 21, 2008===<br />
====Nathan Puhl====<br />
Subcultured single colony of re-streaked transformants in LB + amp and shaker incubated overnight at 37 C</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/ProjectTeam:University of Lethbridge/Project2008-07-15T17:54:16Z<p>Nathan.puhl: /* IRES */</p>
<hr />
<div>__NOTOC__<br />
<br />
<div style="background-color:#000000; color:white"><br />
<br />
{| align="center" style="background-color:#000000; color:white"<br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
!align="center"|<span class="plainlinks" height:3px ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
</span><br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
|}<br />
<br />
<br />
<span style="background-color:#000000; color:white"><!--__TOC__--></span><br />
<br />
==<span style="background-color:#000000; color:white"> '''The “Bacuum" Cleaner – an intelligent self-propelling keener cleaner'''</span> ==<br />
<br />
<!--- The Mission, Experiments ---><br />
Our goal is to create a modified ''Escherichia coli'' bacterium capable of seeking out and degrading toxic aromatic pollutants created during the oil refinery and mining processes. Our “Bacuum" cleaner will respond to this destructive compound through interaction with a programmed riboswitch (an mRNA capable of binding a target molecule with its 5’ UTR which causes a conformational change, thereby regulating any downstream protein translation). By using riboswitches that switch at varying concentrations of target ligand, we can alter the signal which is induced. At low concentrations, we intend to have our riboswitch express the motility protein cheZ in ''E. coli'', thus directing the bacterium towards higher concentrations of our target molecule (i.e. positive chemotaxis). Once it reaches a threshold concentration, a catabolic pathway capable of degrading our target pollutant will be activated.<br />
<br />
We plan to use [http://en.wikipedia.org/wiki/Systematic_Evolution_of_Ligands_by_Exponential_Enrichment <font color=yellow>SELEX</font>] to reprogram the theophylline riboswitch initially characterized by our team last year, aiming to have it bind one of many toxic aromatic compounds. This not only builds on our past work with riboswitches but also demonstrates an alternative function for riboswitch-controlled chemotaxis, changing from a simple detection to active “search and destroy” role. Fluorescent protein expression will be used to demonstrate and characterize the functionality of the riboswitch.<br />
<br />
Our project also has major environmental implications, especially in Alberta, where the oil industry is the driving economic sector. The tailings ponds used to house discarded mining refuse from the oil refineries pose a major environmental dilemma. The problem arises with being unable to collect and degrade the compounds which make up these toxic soups. These not only toxic but often corrosive water beds not only affect the local environment but can also cause severe repercussions to other ecosystems as much of the wildlife relies on getting their food and water from the now disrupted bionetwork. These tailings ponds are often laced with heavy metals and dangerous chemicals, including these toxic aromatic hydrocarbons which can be extremely difficult to breakdown. Despite the efforts of the corporations responsible, some of these chemicals can leech into and contaminate the surrounding ground water and soil. It is our intention that our “Bacuum” will not only remove these compounds from such an aqueous system, but will also eliminate them. This goes one step further than the ordinary less-intelligent bag-dependent vacuum cleaners. Ultimately we aim to have our bacteria convert the target aromatic into a useable compound. One example would be the biosynthesis of fatty acids as an alternative fuel source, creating a potential “Bioreactor” which is capable of creating a more environmentally friendly fuel source.<br />
<br />
== <span style="background-color:#000000; color:white">Project Details</span>==<br />
<br />
<br />
<br />
<br />
<br />
=== <span style="background-color:#000000; color:white">Chemotaxis - the "search" from "search and destroy" </span>===<br />
<br />
Our subproject is to ensure our 'bacuum cleaner' will search out and move towards our potential target. Using a riboswitch which responds to this target, a gene involved in motility in ''Escherichia coli'' (''CheZ'') will be switched to an 'on' state. ''CheZ'' controls bacterial movement by dephosphorylating ''CheY'', altering the bacteria from random tumbling to directed movement. By doing so, we hope to observe the bacteria moving towards the target molecule in a positive chemotaxis manner. <br />
<br />
Because we wish to eventually have our bacteria degrade this activating compound, we need to have a multi-level response system established. Our bacteria should respond to low levels of the target by activating our ''CheZ'' riboswitch (through a strong binding aptamer) and to high levels by activating the catabolic pathway responsible for its degradation. By doing so, we will have created our 'search and destroy' system.<br />
<br />
Our subgroup will attempt to establish a working motility assay to prove control of chemotaxis is possible with an already characterized theophylline-binding riboswitch and later with our new target molecule. We will also use a colour read-out system of two fluorescent proteins to demonstrate the differential binding of the ligand at varying concentrations.<br />
<br />
=== <span style="background-color:#000000; color:white">Riboswitch Characterization</span>===<br />
<br />
This subproject was created to quantify and characterize the binding affinity of the theophylline riboswitch to theophylline as well as other similar aromatic ligands. We will use Green Fluorescent Protein (GFP) sequenced behind the riboswitch to help in this endeavour. When the target ligand is bound to the riboswitch, the production of GFP will be induced, producing green colonies. When the ligand is not bound, GFP will not be produced. We intend to try to characterize the riboswitch with and without the iGEM scar to determine if this produces any effect on the relative effeciency of the riboswitch on the protein.<br />
<br />
We will use variable concentrations of Theophylline, Caffeine and 3-methylxanthine to help with the characterization of the initial theophylline riboswitch. We will also test other aromatic compounds to determine wether or not, the theophylline riboswitch can be utilized to bind them as well. <br />
<br />
Once we have successfully produced mutant riboswitches through SELEX, will we then perform these same characterization experiments to determine their binding affinities.<br />
<br />
[[Image:riboswitch.jpeg|650px|center]]<br />
<br />
===<span style="background-color:#000000; color:white"> SELEX </span>===<br />
<br />
=== <span style="background-color:#000000; color:white">TIR </span>===<br />
<br />
Translation initiation regions (TIRs) are elements found in certain eukaryotic mRNAs that facilitate translation initiation by noncanonical, end independent interactions. We are going to incorporate the TIR from the rpsA gene, encoding ribosomal protein S1, of E. coli into our Bacuum cleaner with green fluoescent protein as a means of monitoring the location of our cells using minimal cell resources. <br />
<br />
The TIR from the rpsA gene will be characterized through the comparison of translation rates for GFP to other canonical RBS. This will allow us to standardize the number of ribosomal initiations per second (RiPS). In generating these parameters we will be able to contribute to building a database of standard parts that can be used to generate biological systems that have a greater degree of reliablity.<br />
<br />
== <span style="background-color:#000000; color:white">Results </span>==<br />
<br />
<br />
----<br />
<br />
<br />
{| align="center" style="background-color:#000000; color:white"<br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
!align="center"|<span class="plainlinks" height:3px ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
</span><br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
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|}<br />
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</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/NotebookTeam:University of Lethbridge/Notebook2008-07-15T17:49:38Z<p>Nathan.puhl: /* "IRES" */</p>
<hr />
<div>__NOTOC__<br />
<br />
<div style="background-color:#000000; color:white"><br />
<br />
{| align="center" style="background-color:#000000; color:white"<br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
!align="center"|<span class="plainlinks" height:3px ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
</span><br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
|}<br />
<br />
Here we have our electronic lab notebooks. <br />
<br />
==<span style="background-color:#000000; color:white">General Lab</span>==<br />
[[Team:University_of_Lethbridge/Notebook/GeneralLabMay|<font color=yellow>May Lab Book</font>]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/GeneralLabJune|<font color=yellow>June Lab Book</font>]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/GeneralLabJuly|<font color=yellow>July Lab Book</font>]]<br />
<br />
==<span style="background-color:#000000; color:white">Chemotaxis</span>==<br />
[[Team:University_of_Lethbridge/Notebook/Project1May|May Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project1June|<font color=yellow>June Lab Book</font>]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project1July|<font color=yellow>July Lab Book</font>]]<br />
<br />
==<span style="background-color:#000000; color:white">Riboswitch/SELEX</span>==<br />
[[Team:University_of_Lethbridge/Notebook/Project2May|May Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project2June|June Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project2July|July Lab Book]]<br />
<br />
==<span style="background-color:#000000; color:white">TIR</span>==<br />
[[Team:University_of_Lethbridge/Notebook/Project3May|May Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project3June|June Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project3July|July Lab Book]]<br />
<br />
==<span style="background-color:#000000; color:white">Metabolic Pathway</span>==<br />
[[Team:University_of_Lethbridge/Notebook/Project4May|May Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project4June|June Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project4July|July Lab Book]]<br />
<br />
<br />
----<br />
<br />
<br />
{| align="center" style="background-color:#000000; color:white"<br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
!align="center"|<span class="plainlinks" height:3px ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
</span><br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
|}<br />
<br />
<br />
<br />
</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/NotebookTeam:University of Lethbridge/Notebook2008-07-15T17:49:25Z<p>Nathan.puhl: /* Riboswitch */</p>
<hr />
<div>__NOTOC__<br />
<br />
<div style="background-color:#000000; color:white"><br />
<br />
{| align="center" style="background-color:#000000; color:white"<br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
!align="center"|<span class="plainlinks" height:3px ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
</span><br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
|}<br />
<br />
Here we have our electronic lab notebooks. <br />
<br />
==<span style="background-color:#000000; color:white">General Lab</span>==<br />
[[Team:University_of_Lethbridge/Notebook/GeneralLabMay|<font color=yellow>May Lab Book</font>]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/GeneralLabJune|<font color=yellow>June Lab Book</font>]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/GeneralLabJuly|<font color=yellow>July Lab Book</font>]]<br />
<br />
==<span style="background-color:#000000; color:white">Chemotaxis</span>==<br />
[[Team:University_of_Lethbridge/Notebook/Project1May|May Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project1June|<font color=yellow>June Lab Book</font>]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project1July|<font color=yellow>July Lab Book</font>]]<br />
<br />
==<span style="background-color:#000000; color:white">Riboswitch/SELEX</span>==<br />
[[Team:University_of_Lethbridge/Notebook/Project2May|May Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project2June|June Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project2July|July Lab Book]]<br />
<br />
==<span style="background-color:#000000; color:white">"IRES"</span>==<br />
[[Team:University_of_Lethbridge/Notebook/Project3May|May Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project3June|June Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project3July|July Lab Book]]<br />
<br />
==<span style="background-color:#000000; color:white">Metabolic Pathway</span>==<br />
[[Team:University_of_Lethbridge/Notebook/Project4May|May Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project4June|June Lab Book]]<br />
<br />
[[Team:University_of_Lethbridge/Notebook/Project4July|July Lab Book]]<br />
<br />
<br />
----<br />
<br />
<br />
{| align="center" style="background-color:#000000; color:white"<br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
!align="center"|<span class="plainlinks" height:3px ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
</span><br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
|}<br />
<br />
<br />
<br />
</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJulyTeam:University of Lethbridge/Notebook/GeneralLabJuly2008-07-15T01:45:25Z<p>Nathan.puhl: </p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Phillips, Andrew====<br />
Made 500 mL of LB agar + amp and 500 mL of Liquid LB<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Alix, Sebastian, Munima, Roxanne, Christa====<br />
<br />
Transformed [http://partsregistry.org/wiki/index.php?title=Part:BBa_J24679 BBa_J24679] (RBS + LacI), [http://partsregistry.org/wiki/index.php?title=Part:BBa_P0440 BBa_P0440] (RBS+TetR+T10+T12), leftover DNA from Double T (June 26, 2008), and 1 uL of pSB1A7 plasmid (June 18, 2008).<br />
<br />
Protocol changes: <br />
-3 uL of DNA<br />
-spin down 200 uL of cells and resuspend in 100 uL of LB; plate on LB + amp<br />
<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa, Alix, Roxanne, Sebastian====<br />
Checked transformation plates. Only the positive control (pSB1A7) had colonies (~1500) indicating that there is nothing wrong with the transformation protocol or cells so we must be having problems with the DNA extraction from the filter paper. Next week we will attempt various changes to the protocol to extract more DNA.<br />
<br />
<br />
===July 8, 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
Made 500mL of LB semi-solid media and poured 24 plates. <br />
Stored in the iGEM 4 C fridge<br />
<br />
====Nathan Puhl, Alix====<br />
Flourescent Reporter<br />
<br />
Transformation from BioBricks LacI (BBA_J24679), TetR (BBa_P0440) and DT (BBa_B0015).<br />
<br />
Protocol:<br />
-punched out 2 spots of filter paper<br />
-15uL TE, for 30 min. @ 50 C<br />
-centrifuge 3 min. @ 15000 g<br />
-freeze 5 min. in -20 C freezer<br />
-heat shock 1 min. in 42 C water bath<br />
-centrifuge 3 min.<br />
-2 uL of plasmid into 25 uL DH5alpha<br />
-leave on ice for 30 min. (Left remaining DT, TetR, LacI to sit overnight at room temp. to possibly test for better DNA recovery)<br />
-put in 42 C water bath for 45 sec.<br />
-chill on ice for 2 min.<br />
-add 1 mL of SOC broth<br />
-incubate cells @ 37 C, 225 rpm for 60 min.<br />
-spin down 400 uL cells (1 min. 16000xG), remove 300 uL<br />
-resuspend<br />
-plate, incubate at 37 C overnight<br />
<br />
Subcultured 3 biobricks (for flourescent reporter) glycerol stocked from last year into 5 mL liquid LB + Amp.<br />
-RFP Sub. (BBa_I13507)<br />
-pLACI (BBa_R0011)<br />
-pSTRONG (BBa_J23119)<br />
<br />
loading dye [http://openwetware.org/wiki/Agarose_gel_loading_dye sizes]</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJulyTeam:University of Lethbridge/Notebook/GeneralLabJuly2008-07-09T02:55:24Z<p>Nathan.puhl: </p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Phillips, Andrew====<br />
Made 500 mL of LB agar + amp and 500 mL of Liquid LB<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Alix, Sebastian, Munima, Roxanne, Christa====<br />
<br />
Transformed [http://partsregistry.org/wiki/index.php?title=Part:BBa_J24679 BBa_J24679] (RBS + LacI), [http://partsregistry.org/wiki/index.php?title=Part:BBa_P0440 BBa_P0440] (RBS+TetR+T10+T12), leftover DNA from Double T (June 26, 2008), and 1 uL of pSB1A7 plasmid (June 18, 2008).<br />
<br />
Protocol changes: <br />
-3 uL of DNA<br />
-spin down 200 uL of cells and resuspend in 100 uL of LB; plate on LB + amp<br />
<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa, Alix, Roxanne, Sebastian====<br />
Checked transformation plates. Only the positive control (pSB1A7) had colonies (~1500) indicating that there is nothing wrong with the transformation protocol or cells so we must be having problems with the DNA extraction from the filter paper. Next week we will attempt various changes to the protocol to extract more DNA.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJuneTeam:University of Lethbridge/Notebook/GeneralLabJune2008-07-09T02:52:48Z<p>Nathan.puhl: </p>
<hr />
<div>===June 6 2008===<br />
====Sebastian, John and Roxanne ====<br />
Prepared 1L of semi-solid media following the procedure found on OpenWetWare.<br />
-10g peptone (substituted for tryptone)<br />
-10g Agar<br />
-10g NaCl<br />
-5g Yeast Extract<br />
<br />
Stored media in fridge.<br />
<br />
<br />
===June 10 2008===<br />
====Christa, Munima, Roxanne, and Sebastian====<br />
Prepared 1L of liquid media following the procedure found on OpenWetWare.<br />
-10g peptone (substituted for tryptone)<br />
-10g NaCl<br />
-5g Yeast Extract<br />
<br />
Poured 36 culture test tubes, and the remainder was left in a 1L Erlenmeyer flask. Stored both in fridge.<br />
<br />
====Christa====<br />
Made an inventory of iGEM 2007 parts in Wieden -80 freezer<br />
[[inventory.xls]]<br />
<br />
====Roxanne====<br />
Defrosted the -20 freezer in the teaching lab for iGEM use (with a little help from my friends!)<br />
<br />
<br />
===June 11 2008===<br />
<br />
====Sebastian, Munima, Roxanne, Christa====<br />
Poured 8 minimal media (labeled control - with blue sharpie) plates and 14 amp plates (labeled amp - with red sharpie)<br />
stored in the 4 C fridge. Amp concentration is always 50ug/mL.<br />
<br />
<br />
===June 16 2008===<br />
====Nathan Puhl, Munima, Christa, Sebastian, Roxanne====<br />
Transformed supercompetent cells with basic biobrick vector [http://partsregistry.org/wiki/index.php?title=Part:pSB1A7 pSB1A7] (ampicillin resistance).<br />
-50 uL of DH5-alpha cells, 0.85 uL 2-mercaptoethanol, 1 uL of plasmid dissolved with 15 uL of ddH2O<br />
-30 min on ice<br />
-45 s at 42 C<br />
-2 min on ice<br />
-Add 0.9 mL of LB shaker incubate at 225 RPM and 37 C<br />
<br />
<br />
===June 17 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
Checked plates<br />
-Only one colony from transformation, not very good efficiency, don't know why because too many possibilities, most <br />
likely amount of DNA due to inability to quantify plasmid from iGEM plates<br />
-Subcultured colony in liquid LB + amp<br />
-Plate 200 uL on LB + amp at 37 C overnight<br />
<br />
<br />
===June 18 2008===<br />
====Munima, Christa, Alix, Nathan Puhl====<br />
Made glycerol stock of pSB1A7 transformed E. coli<br />
<br />
Extracted plasmid from transformed E. coli using the Eppendorf FastPlasmid minikit and stored 4 aliquots of 25 uL in the -20 C freezer.<br />
<br />
<br />
===June 19 2008===<br />
====Nathan Puhl, Alix, Munima, Christa, Roxanne====<br />
Ran plasmids on 1% agarose gel with High range ladder<br />
<br />
[[Image:pSB1A7 plasmid.jpg|500 px]]<br />
<br />
plasmid is ~15 ng/uL<br />
<br />
<br />
===June 24 2008===<br />
====Nathan Puhl, Alix====<br />
Streaked [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522 BBa_I13522] (TetR repressed GFP) onto LB + amp from last year's glycerol stock.<br />
<br />
<br />
===June 25, 2008===<br />
====Nathan Puhl, Sebastian, Alix====<br />
Subcultured BBa_I13522 into liquid LB + amp for plasmid mini prep<br />
<br />
<br />
===June 26, 2008===<br />
====Nathan Puhl, Sebastian, Alix====<br />
Transformed [http://partsregistry.org/wiki/index.php?title=Part:BBa_J5526 BBa_J5526] (RFP complete), [http://partsregistry.org/wiki/index.php?title=Part:BBa_I730002 BBa_I730002] (pLACI-|TetR), and [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 BBa_B0015] (Double T). <br />
<br />
Plasmid mini-prepped GFP complete (BBa_I13522).<br />
<br />
<br />
===June 27, 2008===<br />
====Nathan Puhl, Alix, Munima====<br />
No colonies on any plates. Will try again next week.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_LethbridgeTeam:University of Lethbridge2008-07-04T05:21:24Z<p>Nathan.puhl: /* Our Project: "Bacuum" Cleaner */</p>
<hr />
<div><div style="background-color:#000000; color:white"><br />
<br />
{| align="center" style="background-color:#000000; color:white"<br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
!align="center"|<span class="plainlinks" height:3px ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
</span><br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
|}<br />
<br />
==<span style="background-color:#000000; color:white">About Us</span>==<br />
A nice little blurb about us<br />
<br />
<br />
==<span style="background-color:#000000; color:white">Our Project: "Bacuum" Cleaner</span>==<br />
<br />
[[Image:UofLteamlogo.jpg|500px|right]]<br />
<br />
The tailing ponds created when water is used to extract oil from the Alberta tar sands or other mining operations pose a major environmental dilemma. How can we isolate the tailings and byproducts of the extraction process? Despite the efforts of the corporations responsible, chemicals can leech from the ponds into the surrounding ground water and soil. These toxic and often corrosive water beds not only affect the immediate land and water environment but also cause severe repercussions to larger ecosystems as much of the wildlife relies on getting their food and water from the now disrupted bionetwork. The exact composition of tailing ponds is highly complex, often laced with heavy metals and other toxic chemicals, such as aromatic hydrocarbons, and are difficult and expensive to filter or degrade. One powerful example is the high level of mercury in our fresh water fish, stemming from the tailing ponds of gold mining operations and the refinement of chloralkali metals.<br />
<br />
It is our goal to create a bacterial vacuum cleaner, or “Bacuum”, which will seek out and degrade a particular harmful hydrocarbon. We intend to use the theophylline riboswitch we worked on last year as a platform for directed evolution to create a novel riboswitch capable of binding a particular aromatic hydrocarbon or class of hydrocarbons. The riboswitch will be used to control expression of the motility protein cheZ in ''Escherichia coli'' at low concentrations, thus directing the bacterium towards local areas of high ligand concentration. At a threshold level, a second riboswitch will be used to activate a pathway capable of metabolizing the aromatic hydrocarbons. Ultimately, the resulting product will be funneled into an anabolic pathway, such as the TCA cycle or fatty acid biosynthesis, from which an alternative fuel source can be produced, thereby turning toxic tailings ponds into a bioreactor with the help of a self-guiding "search and destroy" bacterial operative.<br />
<br />
<br />
<br />
[http://parts.mit.edu/igem07/index.php/Lethbridge <font color=yellow>See last year's team page!</font>]<br />
----<br />
<br />
{| align="center" style="background-color:#000000; color:white"<br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
!align="center"|<span class="plainlinks" height:3px ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
</span><br />
<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
</span><br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
|}<br />
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</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/Project1JulyTeam:University of Lethbridge/Notebook/Project1July2008-07-04T05:19:47Z<p>Nathan.puhl: /* Nathan Puhl, Munima, Christa */</p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Puhl====<br />
Subcultured RP1616 into liquid LB and pTopp cells into liquid LB + amp<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Christa, Munima====<br />
Plasma mini prepped pTopp cells<br />
<br />
Attempted to make RP1616 cells competent using the open wet ware [http://openwetware.org/wiki/Preparing_chemically_competent_cells protocol]. 100 uL aliquots of cells were froxen in liquid nitrogen and stored in the -80 freezer.<br />
<br />
Ran CheZ in pUC19 plasmid and pUC19 plasmid from last year with high range ladder. The pUC19 plasmid seems weird, I don't think it should have two bands; I will have to ask someone about that. The CheZ plasmid appears to be the right size (2600 + 620 = 3220 bp)<br />
<br />
[[Image:puc19 + cheZ.jpg|500 px]]<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa====<br />
No colonies on either plate, but the max-efficiency DH5-alpha cells transformed with pSB1A7 were successfully transformed (~1500 cfu). There are two possibilities: 1. the cells are competent but are low efficiency and we did not add enough DNA, or 2. The cells are not competent. To assess possibility one we will try more DNA. If that does not work we will try a protocol using electroporation.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/Project1JulyTeam:University of Lethbridge/Notebook/Project1July2008-07-04T05:19:07Z<p>Nathan.puhl: </p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Puhl====<br />
Subcultured RP1616 into liquid LB and pTopp cells into liquid LB + amp<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Christa, Munima====<br />
Plasma mini prepped pTopp cells<br />
<br />
Attempted to make RP1616 cells competent using the open wet ware [http://openwetware.org/wiki/Preparing_chemically_competent_cells protocol]. 100 uL aliquots of cells were froxen in liquid nitrogen and stored in the -80 freezer.<br />
<br />
Ran CheZ in pUC19 plasmid and pUC19 plasmid from last year with high range ladder. The pUC19 plasmid seems weird, I don't think it should have two bands; I will have to ask someone about that. The CheZ plasmid appears to be the right size (2600 + 620 = 3220 bp)<br />
<br />
[[Image:puc19 + cheZ.jpg|500 px]]<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa====<br />
No colonies on either plate but the DH5-alpha cells transformed with pSB1A7 were successfully transformed (~1500 cfu). There are two possibilities: 1. teh cells are compotent but are low efficiency and we did not add enough DNA, or 2. The cells are not compotent. To assess possibility one we will try more DNA. If that does not work we will try a protocol using electroporation.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJulyTeam:University of Lethbridge/Notebook/GeneralLabJuly2008-07-04T05:14:32Z<p>Nathan.puhl: </p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Puhl, Andrew====<br />
Made 500 mL of LB agar + amp and 500 mL of Liquid LB<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Alix, Sebastian, Munima, Roxanne, Christa====<br />
<br />
Trasformed BBa_J24679( RBS + LacI), BBa_P0440 (RBS+TetR+T10+T12), leftover DNA from Double T (June 26, 2008), and 1 uL of pSB1A7 plasmid (June 18).<br />
<br />
Protocol changes: <br />
-3 uL of DNA<br />
-spin down 200 uL of cells and resuspend in 100 uL of LB; plate on LB + amp<br />
<br />
===July 3, 2008===<br />
====Nathan Puhl, Munima, Christa, Alix, Roxanne, Sebastian====<br />
Checked transformation plates. Only the positive control (pSB1A7) had colonies (~1500) indicating that there is nothing wrong with the transformation protocol or cells so we must be having problems with the DNA extraction from the filter paper. Next week we will attempt various changes to the protocol to extract more DNA.</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/GeneralLabJulyTeam:University of Lethbridge/Notebook/GeneralLabJuly2008-07-04T05:11:57Z<p>Nathan.puhl: /* Nathan, Alix, Sebastian, Munima, Roxanne, Christa */</p>
<hr />
<div>===July 1, 2008===<br />
====Nathan Puhl, Andrew====<br />
Made 500 mL of LB agar + amp and 500 mL of Liquid LB<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Alix, Sebastian, Munima, Roxanne, Christa====<br />
<br />
Trasformed BBa_J24679( RBS + LacI), BBa_P0440 (RBS+TetR+T10+T12), leftover DNA from Double T (June 26, 2008), and 1 uL of pSB1A7 plasmid (June 18).<br />
<br />
Protocol changes: <br />
-3 uL of DNA<br />
-spin down 200 uL of cells and resuspend in 100 uL of LB; plate on LB + amp</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/ProjectTeam:University of Lethbridge/Project2008-07-04T05:11:06Z<p>Nathan.puhl: /* Part 3 */</p>
<hr />
<div>__NOTOC__<br />
<br />
<div style="background-color:#000000; color:white"><br />
<br />
{| align="center" style="background-color:#000000; color:white"<br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
!align="center"|<span class="plainlinks" height:3px ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
</span><br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
|}<br />
<br />
<br />
<span style="background-color:#000000; color:white"><!--__TOC__--></span><br />
<br />
==<span style="background-color:#000000; color:white"> '''The “Bacuum" Cleaner – an intelligent self-propelling keener cleaner'''</span> ==<br />
<br />
<!--- The Mission, Experiments ---><br />
Our goal is to create a modified ''Escherichia coli'' bacterium capable of seeking out and degrading toxic aromatic pollutants created during the oil refinery and mining processes. Our “Bacuum" cleaner will respond to this destructive compound through interaction with a programmed riboswitch (an mRNA capable of binding a target molecule with its 5’ UTR which causes a conformational change, thereby regulating any downstream protein translation). By using riboswitches that switch at varying concentrations of target ligand, we can alter the signal which is induced. At low concentrations, we intend to have our riboswitch express the motility protein cheZ in ''E. coli'', thus directing the bacterium towards higher concentrations of our target molecule (i.e. positive chemotaxis). Once it reaches a threshold concentration, a catabolic pathway capable of degrading our target pollutant will be activated.<br />
<br />
We plan to use [http://en.wikipedia.org/wiki/Systematic_Evolution_of_Ligands_by_Exponential_Enrichment <font color=yellow>SELEX</font>] to reprogram the theophylline riboswitch initially characterized by our team last year, aiming to have it bind one of many toxic aromatic compounds. This not only builds on our past work with riboswitches but also demonstrates an alternative function for riboswitch-controlled chemotaxis, changing from a simple detection to active “search and destroy” role. Fluorescent protein expression will be used to demonstrate and characterize the functionality of the riboswitch.<br />
<br />
Our project also has major environmental implications, especially in Alberta, where the oil industry is the driving economic sector. The tailings ponds used to house discarded mining refuse from the oil refineries pose a major environmental dilemma. The problem arises with being unable to collect and degrade the compounds which make up these toxic soups. These not only toxic but often corrosive water beds not only affect the local environment but can also cause severe repercussions to other ecosystems as much of the wildlife relies on getting their food and water from the now disrupted bionetwork. These tailings ponds are often laced with heavy metals and dangerous chemicals, including these toxic aromatic hydrocarbons which can be extremely difficult to breakdown. Despite the efforts of the corporations responsible, some of these chemicals can leech into and contaminate the surrounding ground water and soil. It is our intention that our “Bacuum” will not only remove these compounds from such an aqueous system, but will also eliminate them. This goes one step further than the ordinary less-intelligent bag-dependent vacuum cleaners. Ultimately we aim to have our bacteria convert the target aromatic into a useable compound. One example would be the biosynthesis of fatty acids as an alternative fuel source, creating a potential “Bioreactor” which is capable of creating a more environmentally friendly fuel source.<br />
<br />
== <span style="background-color:#000000; color:white">Project Details</span>==<br />
<br />
<br />
<br />
<br />
<br />
=== <span style="background-color:#000000; color:white">Chemotaxis - the "search" from "search and destroy" </span>===<br />
<br />
Our subproject is to ensure our 'bacuum cleaner' will search out and move towards our potential target. Using a riboswitch which responds to this target, a gene involved in motility in ''Escherichia coli'' (''CheZ'') will be switched to an 'on' state. ''CheZ'' controls bacterial movement by dephosphorylating ''CheY'', altering the bacteria from random tumbling to directed movement. By doing so, we hope to observe the bacteria moving towards the target molecule in a positive chemotaxis manner. <br />
<br />
Because we wish to eventually have our bacteria degrade this activating compound, we need to have a multi-level response system established. Our bacteria should respond to low levels of the target by activating our ''CheZ'' riboswitch (through a strong binding aptamer) and to high levels by activating the catabolic pathway responsible for its degradation. By doing so, we will have created our 'search and destroy' system.<br />
<br />
Our subgroup will attempt to establish a working motility assay to prove control of chemotaxis is possible with an already characterized theophylline-binding riboswitch and later with our new target molecule. We will also use a colour read-out system of two fluorescent proteins to demonstrate the differential binding of the ligand at varying concentrations.<br />
<br />
=== <span style="background-color:#000000; color:white">Riboswitch Characterization</span>===<br />
<br />
This subproject was created to quantify and characterize the binding affinity of the theophylline riboswitch to theophylline as well as other similar aromatic ligands. We will use Green Fluorescent Protein (GFP) sequenced behind the riboswitch to help in this endeavour. When the target ligand is bound to the riboswitch, the production of GFP will be induced, producing green colonies. When the ligand is not bound, GFP will not be produced. We intend to try to characterize the riboswitch with and without the iGEM scar to determine if this produces any effect on the relative effeciency of the riboswitch on the protein.<br />
<br />
We will use variable concentrations of Theophylline, Caffeine and 3-methylxanthine to help with the characterization of the initial theophylline riboswitch. We will also test other aromatic compounds to determine wether or not, the theophylline riboswitch can be utilized to bind them as well. <br />
<br />
Once we have successfully produced mutant riboswitches through SELEX, will we then perform these same characterization experiments to determine their binding affinities.<br />
<br />
[[Image:riboswitch.jpeg|650px|center]]<br />
<br />
===<span style="background-color:#000000; color:white"> SELEX </span>===<br />
<br />
=== <span style="background-color:#000000; color:white">Part 4 </span>===<br />
<br />
<br />
<br />
<br />
== <span style="background-color:#000000; color:white">Results </span>==<br />
<br />
<br />
----<br />
<br />
<br />
{| align="center" style="background-color:#000000; color:white"<br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
!align="center"|<span class="plainlinks" height:3px ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
</span><br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
|}<br />
<br />
<br />
<br />
</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/ProjectTeam:University of Lethbridge/Project2008-07-04T05:10:14Z<p>Nathan.puhl: /* Riboswitch Characterization */</p>
<hr />
<div>__NOTOC__<br />
<br />
<div style="background-color:#000000; color:white"><br />
<br />
{| align="center" style="background-color:#000000; color:white"<br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
!align="center"|<span class="plainlinks" height:3px ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
</span><br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
|}<br />
<br />
<br />
<span style="background-color:#000000; color:white"><!--__TOC__--></span><br />
<br />
==<span style="background-color:#000000; color:white"> '''The “Bacuum" Cleaner – an intelligent self-propelling keener cleaner'''</span> ==<br />
<br />
<!--- The Mission, Experiments ---><br />
Our goal is to create a modified ''Escherichia coli'' bacterium capable of seeking out and degrading toxic aromatic pollutants created during the oil refinery and mining processes. Our “Bacuum" cleaner will respond to this destructive compound through interaction with a programmed riboswitch (an mRNA capable of binding a target molecule with its 5’ UTR which causes a conformational change, thereby regulating any downstream protein translation). By using riboswitches that switch at varying concentrations of target ligand, we can alter the signal which is induced. At low concentrations, we intend to have our riboswitch express the motility protein cheZ in ''E. coli'', thus directing the bacterium towards higher concentrations of our target molecule (i.e. positive chemotaxis). Once it reaches a threshold concentration, a catabolic pathway capable of degrading our target pollutant will be activated.<br />
<br />
We plan to use [http://en.wikipedia.org/wiki/Systematic_Evolution_of_Ligands_by_Exponential_Enrichment <font color=yellow>SELEX</font>] to reprogram the theophylline riboswitch initially characterized by our team last year, aiming to have it bind one of many toxic aromatic compounds. This not only builds on our past work with riboswitches but also demonstrates an alternative function for riboswitch-controlled chemotaxis, changing from a simple detection to active “search and destroy” role. Fluorescent protein expression will be used to demonstrate and characterize the functionality of the riboswitch.<br />
<br />
Our project also has major environmental implications, especially in Alberta, where the oil industry is the driving economic sector. The tailings ponds used to house discarded mining refuse from the oil refineries pose a major environmental dilemma. The problem arises with being unable to collect and degrade the compounds which make up these toxic soups. These not only toxic but often corrosive water beds not only affect the local environment but can also cause severe repercussions to other ecosystems as much of the wildlife relies on getting their food and water from the now disrupted bionetwork. These tailings ponds are often laced with heavy metals and dangerous chemicals, including these toxic aromatic hydrocarbons which can be extremely difficult to breakdown. Despite the efforts of the corporations responsible, some of these chemicals can leech into and contaminate the surrounding ground water and soil. It is our intention that our “Bacuum” will not only remove these compounds from such an aqueous system, but will also eliminate them. This goes one step further than the ordinary less-intelligent bag-dependent vacuum cleaners. Ultimately we aim to have our bacteria convert the target aromatic into a useable compound. One example would be the biosynthesis of fatty acids as an alternative fuel source, creating a potential “Bioreactor” which is capable of creating a more environmentally friendly fuel source.<br />
<br />
== <span style="background-color:#000000; color:white">Project Details</span>==<br />
<br />
<br />
<br />
<br />
<br />
=== <span style="background-color:#000000; color:white">Chemotaxis - the "search" from "search and destroy" </span>===<br />
<br />
Our subproject is to ensure our 'bacuum cleaner' will search out and move towards our potential target. Using a riboswitch which responds to this target, a gene involved in motility in ''Escherichia coli'' (''CheZ'') will be switched to an 'on' state. ''CheZ'' controls bacterial movement by dephosphorylating ''CheY'', altering the bacteria from random tumbling to directed movement. By doing so, we hope to observe the bacteria moving towards the target molecule in a positive chemotaxis manner. <br />
<br />
Because we wish to eventually have our bacteria degrade this activating compound, we need to have a multi-level response system established. Our bacteria should respond to low levels of the target by activating our ''CheZ'' riboswitch (through a strong binding aptamer) and to high levels by activating the catabolic pathway responsible for its degradation. By doing so, we will have created our 'search and destroy' system.<br />
<br />
Our subgroup will attempt to establish a working motility assay to prove control of chemotaxis is possible with an already characterized theophylline-binding riboswitch and later with our new target molecule. We will also use a colour read-out system of two fluorescent proteins to demonstrate the differential binding of the ligand at varying concentrations.<br />
<br />
=== <span style="background-color:#000000; color:white">Riboswitch Characterization</span>===<br />
<br />
This subproject was created to quantify and characterize the binding affinity of the theophylline riboswitch to theophylline as well as other similar aromatic ligands. We will use Green Fluorescent Protein (GFP) sequenced behind the riboswitch to help in this endeavour. When the target ligand is bound to the riboswitch, the production of GFP will be induced, producing green colonies. When the ligand is not bound, GFP will not be produced. We intend to try to characterize the riboswitch with and without the iGEM scar to determine if this produces any effect on the relative effeciency of the riboswitch on the protein.<br />
<br />
We will use variable concentrations of Theophylline, Caffeine and 3-methylxanthine to help with the characterization of the initial theophylline riboswitch. We will also test other aromatic compounds to determine wether or not, the theophylline riboswitch can be utilized to bind them as well. <br />
<br />
Once we have successfully produced mutant riboswitches through SELEX, will we then perform these same characterization experiments to determine their binding affinities.<br />
<br />
[[Image:riboswitch.jpeg|650px|center]]<br />
<br />
===<span style="background-color:#000000; color:white"> Part 3 </span>===<br />
<br />
<br />
=== <span style="background-color:#000000; color:white">Part 4 </span>===<br />
<br />
<br />
<br />
<br />
== <span style="background-color:#000000; color:white">Results </span>==<br />
<br />
<br />
----<br />
<br />
<br />
{| align="center" style="background-color:#000000; color:white"<br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
!align="center"|<span class="plainlinks" height:3px ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
</span><br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
|}<br />
<br />
<br />
<br />
</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/ProjectTeam:University of Lethbridge/Project2008-07-03T06:17:50Z<p>Nathan.puhl: /* The “Bacuum" Cleaner – an intelligent self-propelling keener cleaner */</p>
<hr />
<div>__NOTOC__<br />
<br />
<div style="background-color:#000000; color:white"><br />
<br />
{| align="center" style="background-color:#000000; color:white"<br />
<br />
!align="center"|[[Image:UofLteamlogo.jpg|150px]]<br />
<br />
!align="center"|<span class="plainlinks" height:3px ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
</span><br />
<br />
!align="center"|<span class="plainlinks" ><br />
[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
</span><br />
<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
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</span><br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
</span><br />
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<span style="background-color:#000000; color:white"><!--__TOC__--></span><br />
<br />
==<span style="background-color:#000000; color:white"> '''The “Bacuum" Cleaner – an intelligent self-propelling keener cleaner'''</span> ==<br />
<br />
<!--- The Mission, Experiments ---><br />
Our goal is to create a modified ''Escherichia coli'' bacterium capable of seeking out and degrading toxic aromatic pollutants created during the oil refinery and mining processes. Our “Bacuum" cleaner will respond to this destructive compound through interaction with a programmed riboswitch (an mRNA capable of binding a target molecule with its 5’ UTR which causes a conformational change, thereby regulating any downstream protein translation). By using riboswitches that switch at varying concentrations of target ligand, we can alter the signal which is induced. At low concentrations, we intend to have our riboswitch express the motility protein cheZ in ''E. coli'', thus directing the bacterium towards higher concentrations of our target molecule (i.e. positive chemotaxis). Once it reaches a threshold concentration, a catabolic pathway capable of degrading our target pollutant will be activated.<br />
<br />
We plan to use [http://en.wikipedia.org/wiki/Systematic_Evolution_of_Ligands_by_Exponential_Enrichment SELEX] to reprogram the theophylline riboswitch initially characterized by our team last year, aiming to have it bind one of many toxic aromatic compounds. This not only builds on our past work with riboswitches but also demonstrates an alternative function for riboswitch-controlled chemotaxis, changing from a simple detection to active “search and destroy” role. Fluorescent protein expression will be used to demonstrate and characterize the functionality of the riboswitch.<br />
<br />
Our project also has major environmental implications, especially in Alberta, where the oil industry is the driving economic sector. The tailings ponds used to house discarded mining refuse from the oil refineries pose a major environmental dilemma. The problem arises with being unable to collect and degrade the compounds which make up these toxic soups. These not only toxic but often corrosive water beds not only affect the local environment but can also cause severe repercussions to other ecosystems as much of the wildlife relies on getting their food and water from the now disrupted bionetwork. These tailings ponds are often laced with heavy metals and dangerous chemicals, including these toxic aromatic hydrocarbons which can be extremely difficult to breakdown. Despite the efforts of the corporations responsible, some of these chemicals can leech into and contaminate the surrounding ground water and soil. It is our intention that our “Bacuum” will not only remove these compounds from such an aqueous system, but will also eliminate them. This goes one step further than the ordinary less-intelligent bag-dependent vacuum cleaners. Ultimately we aim to have our bacteria convert the target aromatic into a useable compound. One example would be the biosynthesis of fatty acids as an alternative fuel source, creating a potential “Bioreactor” which is capable of creating a more environmentally friendly fuel source.<br />
<br />
== <span style="background-color:#000000; color:white">Project Details</span>==<br />
<br />
<br />
<br />
<br />
<br />
=== <span style="background-color:#000000; color:white">Chemotaxis - the "search" from "search and destroy" </span>===<br />
<br />
Our subproject is to ensure our 'bacuum cleaner' will search out and move towards our potential target. Using a riboswitch which responds to this target, a gene involved in motility in ''Escherichia coli'' (''CheZ'') will be switched to an 'on' state. ''CheZ'' controls bacterial movement by dephosphorylating ''CheY'', altering the bacteria from random tumbling to directed movement. By doing so, we hope to observe the bacteria moving towards the target molecule in a positive chemotaxis manner. <br />
<br />
Because we wish to eventually have our bacteria degrade this activating compound, we need to have a multi-level response system established. Our bacteria should respond to low levels of the target by activating our ''CheZ'' riboswitch (through a strong binding aptamer) and to high levels by activating the catabolic pathway responsible for its degradation. By doing so, we will have created our 'search and destroy' system.<br />
<br />
Our subgroup will attempt to establish a working motility assay to prove control of chemotaxis is possible with an already characterized theophylline-binding riboswitch and later with our new target molecule. We will also use a colour read-out system of two fluorescent proteins to demonstrate the differential binding of the ligand at varying concentrations.<br />
<br />
=== <span style="background-color:#000000; color:white">Riboswitch Characterization</span>===<br />
<br />
This subproject was created to quantify and characterize the binding affinity of the theophylline riboswitch to theophylline as well as other similar aromatic ligands. We will use Green Fluorescent Protein (GFP) sequenced behind the riboswitch to help in this endeavour. When the target ligand is bound to the riboswitch, the production of GFP will be induced, producing green colonies. When the ligand is not bound, GFP will not produced. We intend to try to characterize the riboswitch with and without the iGEM scar to determine if this produces any effect on the relative effeciency of the riboswitch on the protein.<br />
<br />
We will use variable concentrations of Theophylline, Caffeine and 3-methylxanthine to help with the characterization of the initial theophylline riboswitch.<br />
<br />
Once we have successfully produced mutant riboswitches through SELEX, will we then perform these same characterization experiments on the transformed ''E. coli'' cells to characterize their binding affinities as well.<br />
<br />
[[Image:riboswitch.jpeg|650px]]<br />
<br />
===<span style="background-color:#000000; color:white"> Part 3 </span>===<br />
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<br />
=== <span style="background-color:#000000; color:white">Part 4 </span>===<br />
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<br />
== <span style="background-color:#000000; color:white">Results </span>==<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
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</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_LethbridgeTeam:University of Lethbridge2008-07-03T06:13:53Z<p>Nathan.puhl: /* Our Project: "Bacuum" Cleaner */</p>
<hr />
<div><div style="background-color:#000000; color:white"><br />
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{| align="center" style="background-color:#000000; color:white"<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge|Home}} https://static.igem.org/mediawiki/2008/6/65/UofLhomebutton.jpg]<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
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<br />
==<span style="background-color:#000000; color:white">About Us</span>==<br />
A nice little blurb about us<br />
<br />
<br />
==<span style="background-color:#000000; color:white">Our Project: "Bacuum" Cleaner</span>==<br />
<br />
[[Image:UofLteamlogo.jpg|500px|right]]<br />
<br />
The tailing ponds created when water is used to extract oil from the Alberta tar sands or other mining operations pose a major environmental dilemma. How can we isolate the tailings and byproducts of the extraction process? Despite the efforts of the corporations responsible, chemicals can leech from the ponds into the surrounding ground water and soil. These toxic and often corrosive water beds not only affect the immediate land and water environment but also cause severe repercussions to larger ecosystems as much of the wildlife relies on getting their food and water from the now disrupted bionetwork. The exact composition of tailing ponds is highly complex, often laced with heavy metals and other toxic chemicals, such as aromatic hydrocarbons, and are difficult and expensive to filter or degrade. One powerful example is the high level of mercury in our fresh water fish, stemming from the tailing ponds of gold mining operations and the refinement of chloralkali metals.<br />
<br />
It is our goal to create a bacterial vacuum cleaner, or “Bacuum”, which will seek out and degrade a particular harmful hydrocarbon. We intend to use the theophylline riboswitch we worked on last year as a platform for directed evolution to create a novel riboswitch capable of binding a particular aromatic hydrocarbon or class of hydrocarbons. The riboswitch will be used to control expression of the motility protein cheZ in E. coli at low concentrations, thus directing the bacterium towards local areas of high ligand concentration. At a threshold level, a second riboswitch will be used to activate a pathway capable of metabolizing the aromatic hydrocarbons. Ultimatley, the resulting product will be funneled into an anabolic pathway, such as the TCA cycle or fatty acid biosynthesis, from which an alternative fuel source can be produced, thereby turning toxic tailings ponds into a bioreactor with the help of a self-guiding "search and destroy" bacterial operative.<br />
<br />
<br />
<br />
[http://parts.mit.edu/igem07/index.php/Lethbridge See last years team page!]<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Team}} https://static.igem.org/mediawiki/2008/4/41/UofLteambutton.jpg]<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Project}} https://static.igem.org/mediawiki/2008/e/e9/UofLprojectbutton.jpg]<br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Parts}} https://static.igem.org/mediawiki/2008/2/28/UofLpartsbutton.jpg]<br />
</span><br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Modeling}} https://static.igem.org/mediawiki/2008/a/af/UofLmodelingbutton.jpg]<br />
</span><br />
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[{{SERVER}}{{localurl:Team:University_of_Lethbridge/Notebook}} https://static.igem.org/mediawiki/2008/0/0e/UofLnotebookbutton.jpg]<br />
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</div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/Project1JuneTeam:University of Lethbridge/Notebook/Project1June2008-07-03T01:15:13Z<p>Nathan.puhl: /* Nathan Puhl, Christa, Munima */</p>
<hr />
<div>===June 16, 2008===<br />
====Nathan Puhl, Munima, Christa, Sebastian, Roxanne====<br />
Plated cheZ knockout strain from glycerol on LB + amp and Plain LB to assess viability and antibiotic resistance at 37 C overnight.<br />
<br />
===June 17 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
cheZ knockout strain viable on LB, no growth on LB + amp - Good<br />
<br />
===June 30, 2008===<br />
====Nathan Puhl, Christa, Munima====<br />
plated RP1616 and pTopp cells from glycerol stocks onto LB and LB + amp, respectively<br />
<br />
===July 1, 2008===<br />
====Nathan Puhl====<br />
Subcultured RP1616 into liquid LB and pTopp cells into liquid LB + amp<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Christa, Munima====<br />
Plasma mini prepped pTopp cells<br />
<br />
Attempted to make RP1616 cells competent using the open wet ware [http://openwetware.org/wiki/Preparing_chemically_competent_cells protocol]. 100 uL aliquots of cells were froxen in liquid nitrogen and stored in the -80 freezer.<br />
<br />
Ran CheZ in pUC19 plasmid and pUC19 plasmid from last year with high range ladder. The pUC19 plasmid seems weird, I don't think it should have two bands; I will have to ask someone about that. The CheZ plasmid appears to be the right size (2600 + 620 = 3220 bp)<br />
<br />
[[Image:puc19 + cheZ.jpg|500 px]]</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/Project1JuneTeam:University of Lethbridge/Notebook/Project1June2008-07-03T01:13:17Z<p>Nathan.puhl: /* Nathan Puhl, Christa, Munima */</p>
<hr />
<div>===June 16, 2008===<br />
====Nathan Puhl, Munima, Christa, Sebastian, Roxanne====<br />
Plated cheZ knockout strain from glycerol on LB + amp and Plain LB to assess viability and antibiotic resistance at 37 C overnight.<br />
<br />
===June 17 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
cheZ knockout strain viable on LB, no growth on LB + amp - Good<br />
<br />
===June 30, 2008===<br />
====Nathan Puhl, Christa, Munima====<br />
plated RP1616 and pTopp cells from glycerol stocks onto LB and LB + amp, respectively<br />
<br />
===July 1, 2008===<br />
====Nathan Puhl====<br />
Subcultured RP1616 into liquid LB and pTopp cells into liquid LB + amp<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Christa, Munima====<br />
Plasma mini prepped pTopp cells<br />
<br />
Attempted to make RP1616 cells competent using the open wet ware [http://openwetware.org/wiki/Preparing_chemically_competent_cells protocol]. 100 uL aliquots of cells were froxen in liquid nitrogen and stored in the -80 freezer.<br />
<br />
Ran CheZ in pUC19 plasmid and pUC19 plasmid from last year<br />
<br />
[[Image:puc19 + cheZ.jpg|500 px]]</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/Project1JuneTeam:University of Lethbridge/Notebook/Project1June2008-07-03T01:13:06Z<p>Nathan.puhl: </p>
<hr />
<div>===June 16, 2008===<br />
====Nathan Puhl, Munima, Christa, Sebastian, Roxanne====<br />
Plated cheZ knockout strain from glycerol on LB + amp and Plain LB to assess viability and antibiotic resistance at 37 C overnight.<br />
<br />
===June 17 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
cheZ knockout strain viable on LB, no growth on LB + amp - Good<br />
<br />
===June 30, 2008===<br />
====Nathan Puhl, Christa, Munima====<br />
plated RP1616 and pTopp cells from glycerol stocks onto LB and LB + amp, respectively<br />
<br />
===July 1, 2008===<br />
====Nathan Puhl====<br />
Subcultured RP1616 into liquid LB and pTopp cells into liquid LB + amp<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Christa, Munima====<br />
Plasma mini prepped pTopp cells<br />
<br />
Attempted to make RP1616 cells competent using the open wet ware [http://openwetware.org/wiki/Preparing_chemically_competent_cells protocol]. 100 uL aliquots of cells were froxen in liquid nitrogen and stored in the -80 freezer.<br />
<br />
Ran CheZ in pUC19 plasmid and pUC19 plasmid from last year<br />
[[Image:puc19 + cheZ.jpg|500 px]]</div>Nathan.puhlhttp://2008.igem.org/File:Puc19_%2B_cheZ.jpgFile:Puc19 + cheZ.jpg2008-07-03T01:08:51Z<p>Nathan.puhl: </p>
<hr />
<div></div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/Project1JuneTeam:University of Lethbridge/Notebook/Project1June2008-07-03T01:08:33Z<p>Nathan.puhl: </p>
<hr />
<div>===June 16, 2008===<br />
====Nathan Puhl, Munima, Christa, Sebastian, Roxanne====<br />
Plated cheZ knockout strain from glycerol on LB + amp and Plain LB to assess viability and antibiotic resistance at 37 C overnight.<br />
<br />
===June 17 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
cheZ knockout strain viable on LB, no growth on LB + amp - Good<br />
<br />
===June 30, 2008===<br />
====Nathan Puhl, Christa, Munima====<br />
plated RP1616 and pTopp cells from glycerol stocks onto LB and LB + amp, respectively<br />
<br />
===July 1, 2008===<br />
====Nathan Puhl====<br />
Subcultured RP1616 into liquid LB and pTopp cells into liquid LB + amp<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Christa, Munima====<br />
Plasma mini prepped pTopp cells<br />
<br />
Attempted to make RP1616 cells competent using [http://openwetware.org/wiki/Preparing_chemically_competent_cells]. 100 uL aliquots of cells were froxen in liquid nitrogen and stored in the -80 freezer.<br />
<br />
Ran CheZ in pUC19 plasmid and pUC19 plasmid from last year<br />
[[Image:puc19 + cheZ.jpg]]</div>Nathan.puhlhttp://2008.igem.org/Team:University_of_Lethbridge/Notebook/Project1JuneTeam:University of Lethbridge/Notebook/Project1June2008-07-03T01:05:17Z<p>Nathan.puhl: </p>
<hr />
<div>===June 16, 2008===<br />
====Nathan Puhl, Munima, Christa, Sebastian, Roxanne====<br />
Plated cheZ knockout strain from glycerol on LB + amp and Plain LB to assess viability and antibiotic resistance at 37 C overnight.<br />
<br />
===June 17 2008===<br />
====Munima, Christa, Nathan Puhl====<br />
cheZ knockout strain viable on LB, no growth on LB + amp - Good<br />
<br />
===June 30, 2008===<br />
====Nathan Puhl, Christa, Munima====<br />
plated RP1616 and pTopp cells from glycerol stocks onto LB and LB + amp, respectively<br />
<br />
===July 1, 2008===<br />
====Nathan Puhl====<br />
Subcultured RP1616 into liquid LB and pTopp cells into liquid LB + amp<br />
<br />
===July 2, 2008===<br />
====Nathan Puhl, Christa, Munima====<br />
Plasma mini prepped pTopp cells<br />
<br />
Attempted to make RP1616 cells competent using [http://openwetware.org/wiki/Preparing_chemically_competent_cells] 100 uL aliquots of cells were froxen in liquid nitrogen and stored in the -80 freezer.<br />
<br />
Ran CheZ in pUC19 plasmid and pUC19 plasmid from last year</div>Nathan.puhl