http://2008.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Sally7302008.igem.org - User contributions [en]2024-03-29T04:58:47ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T04:27:29Z<p>Sally730: /* Special protocol */</p>
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<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', 105:433–449 (2008), permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== Design Abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal.<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
==== Explanation ====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
=== Effective Additives ===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
=== References for special protocol: ===<br />
1. David E. Birch et al., '''Simplified hot start PCR''', ''Nature'' 381:445-446 (1996)<br />
<br />
2. S.Kaijalainen et al., '''An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose''', ''Nucleic Acids Research'' 21:2959-2960 (1993)<br />
<br />
3. Yukihiro Kitade et al., '''Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta) gene''', ''Journal of Applied Phycology'' 15:555-557 (2003)<br />
<br />
4. Markus Ralser et al., '''An efficient and economic enhancer mix for PCR''', ''Biochemical and Biophysical Research Communications'' 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
===1. Switch on the degradation pathway===<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
===2. Solve the bottleneck in PCBs degradation pathway===<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
===3. Control on the amount of PCBs molecular that enter the cell.===<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
===4. System guarantee===<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
===References for Design Details:===<br />
<br />
[1] Kensuke Furukawa, et al., '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of Bioscience and Bioengineering'' 105:433–449 (2008)<br />
<br />
[2] Stephen Y. K. Seah, et al., '''Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls''', ''Journal of Bacteriology'' 183:1511–1516 (2001)<br />
<br />
[3] Sang-ho Park, et al., '''Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants''', ''Environmental Monitoring and Assessment'' 92:241–251 (2004)<br />
<br />
[4] Qinhong Wang, et al., '''Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery''', ''Biotechnology and Bioengineering'', 98:842-853 (2007)<br />
<br />
[5] Jessika Feliciano, et al., '''ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls''', ''Analytical and Bioanalytical Chemistry'', 385: 807–813 (2006)<br />
<br />
== Results ==<br />
===1. Protocols===<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
===2. Plasmids constructed===<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
===3. A functional part ===<br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector.<br />
<br />
== Acknowledgments ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis, Division of Microbiology, GBF-National Reasearch Centre for Biotechnology, D-38124 Braunschweig, Germany<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* Professor David N. Dowling, Department of Microbiology, University College, Cork, Ireland<br />
<br />
For the bacteria E. coil SMl0 provided.<br />
<br />
* Professor Junfeng Niu, School of Enviroment, Beijing Normal University <br />
<br />
For his instructions and advices<br />
<br />
* Dr Yingwu Huang, Bioinformatics Institute, Tsinghua University<br />
<br />
For his idea and instructions <br />
<br />
* [[Team:Tsinghua|Tsinghua Team]]<br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* [[Team:Chiba|Chiba Team]]<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* [[Team:Tokyo_Tech|Tokyo Tech Team]]<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* [[Team:USTC|USTC Team]]<br />
<br />
For useful suggestions on our experiments and delicious food in Hefei, especially Bo and Jian.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T04:05:47Z<p>Sally730: /* Acknowledgments */</p>
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!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', 105:433–449 (2008), permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== Design Abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal.<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
=== References for special protocol: ===<br />
1. David E. Birch et al., '''Simplified hot start PCR''', ''Nature'' 381:445-446 (1996)<br />
<br />
2. S.Kaijalainen et al., '''An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose''', ''Nucleic Acids Research'' 21:2959-2960 (1993)<br />
<br />
3. Yukihiro Kitade et al., '''Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta) gene''', ''Journal of Applied Phycology'' 15:555-557 (2003)<br />
<br />
4. Markus Ralser et al., '''An efficient and economic enhancer mix for PCR''', ''Biochemical and Biophysical Research Communications'' 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
===1. Switch on the degradation pathway===<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
===2. Solve the bottleneck in PCBs degradation pathway===<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
===3. Control on the amount of PCBs molecular that enter the cell.===<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
===4. System guarantee===<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
===References for Design Details:===<br />
<br />
[1] Kensuke Furukawa, et al., '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of Bioscience and Bioengineering'' 105:433–449 (2008)<br />
<br />
[2] Stephen Y. K. Seah, et al., '''Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls''', ''Journal of Bacteriology'' 183:1511–1516 (2001)<br />
<br />
[3] Sang-ho Park, et al., '''Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants''', ''Environmental Monitoring and Assessment'' 92:241–251 (2004)<br />
<br />
[4] Qinhong Wang, et al., '''Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery''', ''Biotechnology and Bioengineering'', 98:842-853 (2007)<br />
<br />
[5] Jessika Feliciano, et al., '''ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls''', ''Analytical and Bioanalytical Chemistry'', 385: 807–813 (2006)<br />
<br />
== Results ==<br />
===1. Protocols===<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
===2. Plasmids constructed===<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
===3. A functional part ===<br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector.<br />
<br />
== Acknowledgments ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis, Division of Microbiology, GBF-National Reasearch Centre for Biotechnology, D-38124 Braunschweig, Germany<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* Professor David N. Dowling, Department of Microbiology, University College, Cork, Ireland<br />
<br />
For the bacteria E. coil SMl0 provided.<br />
<br />
* Professor Junfeng Niu, School of Enviroment, Beijing Normal University <br />
<br />
For his instructions and advices<br />
<br />
* Dr Yingwu Huang, Bioinformatics Institute, Tsinghua University<br />
<br />
For his idea and instructions <br />
<br />
* [[Team:Tsinghua|Tsinghua Team]]<br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* [[Team:Chiba|Chiba Team]]<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* [[Team:Tokyo_Tech|Tokyo Tech Team]]<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* [[Team:USTC|USTC Team]]<br />
<br />
For useful suggestions on our experiments and delicious food in Hefei, especially Bo and Jian.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/NotebookTeam:Beijing Normal/Notebook2008-10-30T04:02:46Z<p>Sally730: /* Dynamics */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
=='''Dynamics'''==<br />
*== '''Communications''' ==<br />
**17th June: Attend the Asian Workshop meeting.<br />
<br />
**17th July: Meet up with Tsinghua Team, and learn fron their experiences.<br />
<br />
**25th July: With Invitrogen.Co. Ltd and Tsinghua team to sign up a contract.<br />
<br />
**27th July: Have a discussion with Tsinghua team about the transformation efficiency. <br />
We generalize some key words from the discussion: <br />
<br />
1. Make sure the concentration of the plasmids from the biobrick is enough for transformation. <br />
<br />
2. Keep a low temprature when enzyme digestion is performed <br />
<br />
3. The transformation product are always incubated for more than 1h. <br />
<br />
<br />
**9th September: We and Tsinghua team get together in our lab. We have a happy time together and share a lot. <br />
<br />
**10th September: We provide Tsinghua Team with the cells harboring pSB1AC3 plasmids. We did the the tramsformation for them. Because they found their transformation effeciency is relatively low.<br />
<br />
**17th September: Tsinghua Team told us that the vector that we provided proved to work well. We were all pretty happy about this positive result.<br />
<br />
**3th October: We and Chiba Team had a happy conversation via googletalk today. We share all the success and frustrations in the process. And we exchange our opinion about promotor function measurement. They told us that they have used BBa_T9002 because their project uses quorum sensing and the part(T9002) was GFP producer controlled by 3OC6HSL Receiver Device.And their result is pretty satisfying. <br />
<br />
**5th October: We share the experience of measurment with Tokyo Tech University Team. They tell us to use BBa_I13522 (http://partsregistry.org/Part:BBa_I13522) as a positive control when we measure the function of promotors.<br />
<br />
**9th October: We test the founction of Part BBa_I719015. The result indicates that it works well. The cells (BL21(DE3) as host) turn green when induced by IPTG.<br />
<br />
**15th October: Our T-shirt is designed today. It is very pretty. See image below.<br />
<br />
**19th, 20th October: We have a good discussion with USTC team in Hefei and they give us several important suggestions on experiments.<br />
<br />
[[Image:t-shirt.jpg]]<br />
<br />
[[Image:t-shirt1.jpg]]<br />
<br />
'''Our logo is designed finally. It is a visual field through a microscope. You could see us in this microscope.'''<br />
<br />
[[Image:our logo.jpg]]<br />
<br />
** Our iGem is coming to the end. But our projet will continue. <br />
<br />
Of course, we encountered many obstacles and difficulties such as financial problems, cooperation and communication problems of teammates, spirit pressure, time management and so on. But all the hardship turned out to be a precious experience that helped us grow up. Anyway, iGem is one of our most precious experiences and we will never forget these days and night in the lab and meeting room, all the laughs and tears. We have learned a lot in this project except the attending the competition itself. <br />
<br />
<br />
<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=07}}<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=08}}<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=09}}<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=10}}<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=11}}</div>Sally730http://2008.igem.org/File:Team_member_5.pngFile:Team member 5.png2008-10-30T03:58:33Z<p>Sally730: uploaded a new version of "Image:Team member 5.png": Reverted to version as of 00:22, 30 October 2008</p>
<hr />
<div></div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T03:53:58Z<p>Sally730: /* Acknowledgments */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', 105:433–449 (2008), permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== Design Abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal.<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
=== References for special protocol: ===<br />
1. David E. Birch et al., '''Simplified hot start PCR''', ''Nature'' 381:445-446 (1996)<br />
<br />
2. S.Kaijalainen et al., '''An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose''', ''Nucleic Acids Research'' 21:2959-2960 (1993)<br />
<br />
3. Yukihiro Kitade et al., '''Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta) gene''', ''Journal of Applied Phycology'' 15:555-557 (2003)<br />
<br />
4. Markus Ralser et al., '''An efficient and economic enhancer mix for PCR''', ''Biochemical and Biophysical Research Communications'' 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
===1. Switch on the degradation pathway===<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
===2. Solve the bottleneck in PCBs degradation pathway===<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
===3. Control on the amount of PCBs molecular that enter the cell.===<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
===4. System guarantee===<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
===References for Design Details:===<br />
<br />
[1] Kensuke Furukawa, et al., '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of Bioscience and Bioengineering'' 105:433–449 (2008)<br />
<br />
[2] Stephen Y. K. Seah, et al., '''Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls''', ''Journal of Bacteriology'' 183:1511–1516 (2001)<br />
<br />
[3] Sang-ho Park, et al., '''Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants''', ''Environmental Monitoring and Assessment'' 92:241–251 (2004)<br />
<br />
[4] Qinhong Wang, et al., '''Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery''', ''Biotechnology and Bioengineering'', 98:842-853 (2007)<br />
<br />
[5] Jessika Feliciano, et al., '''ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls''', ''Analytical and Bioanalytical Chemistry'', 385: 807–813 (2006)<br />
<br />
== Results ==<br />
===1. Protocols===<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
===2. Plasmids constructed===<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
===3. A functional part ===<br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector.<br />
<br />
== Acknowledgments ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis, Division of Microbiology, GBF-National Reasearch Centre for Biotechnology, D-38124 Braunschweig, Germany<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* Professor David N. Dowling, Department of Microbiology, University College, Cork, Ireland<br />
<br />
For the bacteria E. coil SMl0 provided.<br />
<br />
* Professor Junfeng Niu, School of Enviroment, Beijing Normal University <br />
<br />
For his instructions and advices<br />
<br />
* [[Team:Tsinghua|Tsinghua Team]]<br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* [[Team:Chiba|Chiba Team]]<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* [[Team:Tokyo_Tech|Tokyo Tech Team]]<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* [[Team:USTC|USTC Team]]<br />
<br />
For useful suggestions on our experiments and delicious food in Hefei, especially Bo and Jian.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T03:52:42Z<p>Sally730: /* Acknowledgments */</p>
<hr />
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!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', 105:433–449 (2008), permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== Design Abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal.<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
=== References for special protocol: ===<br />
1. David E. Birch et al., '''Simplified hot start PCR''', ''Nature'' 381:445-446 (1996)<br />
<br />
2. S.Kaijalainen et al., '''An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose''', ''Nucleic Acids Research'' 21:2959-2960 (1993)<br />
<br />
3. Yukihiro Kitade et al., '''Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta) gene''', ''Journal of Applied Phycology'' 15:555-557 (2003)<br />
<br />
4. Markus Ralser et al., '''An efficient and economic enhancer mix for PCR''', ''Biochemical and Biophysical Research Communications'' 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
===1. Switch on the degradation pathway===<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
===2. Solve the bottleneck in PCBs degradation pathway===<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
===3. Control on the amount of PCBs molecular that enter the cell.===<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
===4. System guarantee===<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
===References for Design Details:===<br />
<br />
[1] Kensuke Furukawa, et al., '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of Bioscience and Bioengineering'' 105:433–449 (2008)<br />
<br />
[2] Stephen Y. K. Seah, et al., '''Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls''', ''Journal of Bacteriology'' 183:1511–1516 (2001)<br />
<br />
[3] Sang-ho Park, et al., '''Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants''', ''Environmental Monitoring and Assessment'' 92:241–251 (2004)<br />
<br />
[4] Qinhong Wang, et al., '''Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery''', ''Biotechnology and Bioengineering'', 98:842-853 (2007)<br />
<br />
[5] Jessika Feliciano, et al., '''ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls''', ''Analytical and Bioanalytical Chemistry'', 385: 807–813 (2006)<br />
<br />
== Results ==<br />
===1. Protocols===<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
===2. Plasmids constructed===<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
===3. A functional part ===<br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector.<br />
<br />
== Acknowledgments ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis Division of Microbiology, GBF-National Reasearch Centre for Biotechnology, D-38124 Braunschweig, Germany<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* Professor David N. Dowling, Department of Microbiology, University College, Cork, Ireland<br />
<br />
For the bacteria E. coil SMl0 provided.<br />
<br />
* Professor Junfeng Niu, School of Enviroment, Beijing Normal University <br />
<br />
For his instructions and advices<br />
<br />
* [[Team:Tsinghua|Tsinghua Team]]<br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* [[Team:Chiba|Chiba Team]]<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* [[Team:Tokyo_Tech|Tokyo Tech Team]]<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* [[Team:USTC|USTC Team]]<br />
<br />
For useful suggestions on our experiments and delicious food in Hefei, especially Bo and Jian.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T03:52:20Z<p>Sally730: /* Acknowledgments */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', 105:433–449 (2008), permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== Design Abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal.<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
=== References for special protocol: ===<br />
1. David E. Birch et al., '''Simplified hot start PCR''', ''Nature'' 381:445-446 (1996)<br />
<br />
2. S.Kaijalainen et al., '''An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose''', ''Nucleic Acids Research'' 21:2959-2960 (1993)<br />
<br />
3. Yukihiro Kitade et al., '''Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta) gene''', ''Journal of Applied Phycology'' 15:555-557 (2003)<br />
<br />
4. Markus Ralser et al., '''An efficient and economic enhancer mix for PCR''', ''Biochemical and Biophysical Research Communications'' 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
===1. Switch on the degradation pathway===<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
===2. Solve the bottleneck in PCBs degradation pathway===<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
===3. Control on the amount of PCBs molecular that enter the cell.===<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
===4. System guarantee===<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
===References for Design Details:===<br />
<br />
[1] Kensuke Furukawa, et al., '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of Bioscience and Bioengineering'' 105:433–449 (2008)<br />
<br />
[2] Stephen Y. K. Seah, et al., '''Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls''', ''Journal of Bacteriology'' 183:1511–1516 (2001)<br />
<br />
[3] Sang-ho Park, et al., '''Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants''', ''Environmental Monitoring and Assessment'' 92:241–251 (2004)<br />
<br />
[4] Qinhong Wang, et al., '''Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery''', ''Biotechnology and Bioengineering'', 98:842-853 (2007)<br />
<br />
[5] Jessika Feliciano, et al., '''ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls''', ''Analytical and Bioanalytical Chemistry'', 385: 807–813 (2006)<br />
<br />
== Results ==<br />
===1. Protocols===<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
===2. Plasmids constructed===<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
===3. A functional part ===<br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector.<br />
<br />
== Acknowledgments ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
Division of Microbiology, GBF-National Reasearch Centre for Biotechnology, D-38124 Braunschweig, Germany<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* Professor David N. Dowling, Department of Microbiology, University College, Cork, Ireland<br />
<br />
For the bacteria E. coil SMl0 provided.<br />
<br />
* Professor Junfeng Niu, School of Enviroment, Beijing Normal University <br />
<br />
For his instructions and advices<br />
<br />
* [[Team:Tsinghua|Tsinghua Team]]<br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* [[Team:Chiba|Chiba Team]]<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* [[Team:Tokyo_Tech|Tokyo Tech Team]]<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* [[Team:USTC|USTC Team]]<br />
<br />
For useful suggestions on our experiments and delicious food in Hefei, especially Bo and Jian.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T03:50:57Z<p>Sally730: /* Acknowledgments */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', 105:433–449 (2008), permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== Design Abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal.<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
=== References for special protocol: ===<br />
1. David E. Birch et al., '''Simplified hot start PCR''', ''Nature'' 381:445-446 (1996)<br />
<br />
2. S.Kaijalainen et al., '''An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose''', ''Nucleic Acids Research'' 21:2959-2960 (1993)<br />
<br />
3. Yukihiro Kitade et al., '''Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta) gene''', ''Journal of Applied Phycology'' 15:555-557 (2003)<br />
<br />
4. Markus Ralser et al., '''An efficient and economic enhancer mix for PCR''', ''Biochemical and Biophysical Research Communications'' 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
===1. Switch on the degradation pathway===<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
===2. Solve the bottleneck in PCBs degradation pathway===<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
===3. Control on the amount of PCBs molecular that enter the cell.===<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
===4. System guarantee===<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
===References for Design Details:===<br />
<br />
[1] Kensuke Furukawa, et al., '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of Bioscience and Bioengineering'' 105:433–449 (2008)<br />
<br />
[2] Stephen Y. K. Seah, et al., '''Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls''', ''Journal of Bacteriology'' 183:1511–1516 (2001)<br />
<br />
[3] Sang-ho Park, et al., '''Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants''', ''Environmental Monitoring and Assessment'' 92:241–251 (2004)<br />
<br />
[4] Qinhong Wang, et al., '''Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery''', ''Biotechnology and Bioengineering'', 98:842-853 (2007)<br />
<br />
[5] Jessika Feliciano, et al., '''ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls''', ''Analytical and Bioanalytical Chemistry'', 385: 807–813 (2006)<br />
<br />
== Results ==<br />
===1. Protocols===<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
===2. Plasmids constructed===<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
===3. A functional part ===<br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector.<br />
<br />
== Acknowledgments ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis,<br />
Division of Microbiology, GBF-National Reasearch Centre for Biotechnology, D-38124 Braunschweig, Germany<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* Professor David N. Dowling, Department of Microbiology, University College, Cork, Ireland<br />
<br />
For the bacteria E. coil SMl0 provided.<br />
<br />
* Professor Junfeng Niu, School of Enviroment, Beijing Normal University <br />
<br />
For his instructions and advices<br />
<br />
* [[Team:Tsinghua|Tsinghua Team]]<br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* [[Team:Chiba|Chiba Team]]<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* [[Team:Tokyo_Tech|Tokyo Tech Team]]<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* [[Team:USTC|USTC Team]]<br />
<br />
For useful suggestions on our experiments and delicious food in Hefei, especially Bo and Jian.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T03:48:43Z<p>Sally730: /* Acknowledgments */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', 105:433–449 (2008), permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== Design Abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal.<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
=== References for special protocol: ===<br />
1. David E. Birch et al., '''Simplified hot start PCR''', ''Nature'' 381:445-446 (1996)<br />
<br />
2. S.Kaijalainen et al., '''An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose''', ''Nucleic Acids Research'' 21:2959-2960 (1993)<br />
<br />
3. Yukihiro Kitade et al., '''Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta) gene''', ''Journal of Applied Phycology'' 15:555-557 (2003)<br />
<br />
4. Markus Ralser et al., '''An efficient and economic enhancer mix for PCR''', ''Biochemical and Biophysical Research Communications'' 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
===1. Switch on the degradation pathway===<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
===2. Solve the bottleneck in PCBs degradation pathway===<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
===3. Control on the amount of PCBs molecular that enter the cell.===<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
===4. System guarantee===<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
===References for Design Details:===<br />
<br />
[1] Kensuke Furukawa, et al., '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of Bioscience and Bioengineering'' 105:433–449 (2008)<br />
<br />
[2] Stephen Y. K. Seah, et al., '''Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls''', ''Journal of Bacteriology'' 183:1511–1516 (2001)<br />
<br />
[3] Sang-ho Park, et al., '''Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants''', ''Environmental Monitoring and Assessment'' 92:241–251 (2004)<br />
<br />
[4] Qinhong Wang, et al., '''Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery''', ''Biotechnology and Bioengineering'', 98:842-853 (2007)<br />
<br />
[5] Jessika Feliciano, et al., '''ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls''', ''Analytical and Bioanalytical Chemistry'', 385: 807–813 (2006)<br />
<br />
== Results ==<br />
===1. Protocols===<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
===2. Plasmids constructed===<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
===3. A functional part ===<br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector.<br />
<br />
== Acknowledgments ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis, Division of Microbiology, GBF-National Reasearch Centre for Biotechnology, D-38124 Braunschweig, Germany<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* Professor David N. Dowling, Department of Microbiology, University College, Cork, Ireland<br />
<br />
For the bacteria E. coil SMl0.<br />
<br />
* Professor Junfeng Niu, School of Enviroment, Beijing Normal University <br />
<br />
For his instructions and advices<br />
<br />
* [[Team:Tsinghua|Tsinghua Team]]<br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* [[Team:Chiba|Chiba Team]]<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* [[Team:Tokyo_Tech|Tokyo Tech Team]]<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* [[Team:USTC|USTC Team]]<br />
<br />
For suggestions on our experiments and delicious food in Hefei.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T03:45:51Z<p>Sally730: /* Acknowledgments */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', 105:433–449 (2008), permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== Design Abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal.<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
=== References for special protocol: ===<br />
1. David E. Birch et al., '''Simplified hot start PCR''', ''Nature'' 381:445-446 (1996)<br />
<br />
2. S.Kaijalainen et al., '''An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose''', ''Nucleic Acids Research'' 21:2959-2960 (1993)<br />
<br />
3. Yukihiro Kitade et al., '''Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta) gene''', ''Journal of Applied Phycology'' 15:555-557 (2003)<br />
<br />
4. Markus Ralser et al., '''An efficient and economic enhancer mix for PCR''', ''Biochemical and Biophysical Research Communications'' 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
===1. Switch on the degradation pathway===<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
===2. Solve the bottleneck in PCBs degradation pathway===<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
===3. Control on the amount of PCBs molecular that enter the cell.===<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
===4. System guarantee===<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
===References for Design Details:===<br />
<br />
[1] Kensuke Furukawa, et al., '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of Bioscience and Bioengineering'' 105:433–449 (2008)<br />
<br />
[2] Stephen Y. K. Seah, et al., '''Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls''', ''Journal of Bacteriology'' 183:1511–1516 (2001)<br />
<br />
[3] Sang-ho Park, et al., '''Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants''', ''Environmental Monitoring and Assessment'' 92:241–251 (2004)<br />
<br />
[4] Qinhong Wang, et al., '''Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery''', ''Biotechnology and Bioengineering'', 98:842-853 (2007)<br />
<br />
[5] Jessika Feliciano, et al., '''ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls''', ''Analytical and Bioanalytical Chemistry'', 385: 807–813 (2006)<br />
<br />
== Results ==<br />
===1. Protocols===<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
===2. Plasmids constructed===<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
===3. A functional part ===<br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector.<br />
<br />
== Acknowledgments ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* Professor David N. Dowling, Department of Microbiology, University College, Cork, Ireland<br />
<br />
For the bacteria E. coil SMl0.<br />
<br />
* Professor Junfeng Niu, School of Enviroment, Beijing Normal University <br />
<br />
For his instructions and advices<br />
<br />
* [[Team:Tsinghua|Tsinghua Team]]<br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* [[Team:Chiba|Chiba Team]]<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* [[Team:Tokyo_Tech|Tokyo Tech Team]]<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* [[Team:USTC|USTC Team]]<br />
<br />
For suggestions on our experiments and delicious food in Hefei.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T03:45:16Z<p>Sally730: /* Acknowledgments */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', 105:433–449 (2008), permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== Design Abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal.<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
=== References for special protocol: ===<br />
1. David E. Birch et al., '''Simplified hot start PCR''', ''Nature'' 381:445-446 (1996)<br />
<br />
2. S.Kaijalainen et al., '''An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose''', ''Nucleic Acids Research'' 21:2959-2960 (1993)<br />
<br />
3. Yukihiro Kitade et al., '''Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta) gene''', ''Journal of Applied Phycology'' 15:555-557 (2003)<br />
<br />
4. Markus Ralser et al., '''An efficient and economic enhancer mix for PCR''', ''Biochemical and Biophysical Research Communications'' 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
===1. Switch on the degradation pathway===<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
===2. Solve the bottleneck in PCBs degradation pathway===<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
===3. Control on the amount of PCBs molecular that enter the cell.===<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
===4. System guarantee===<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
===References for Design Details:===<br />
<br />
[1] Kensuke Furukawa, et al., '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of Bioscience and Bioengineering'' 105:433–449 (2008)<br />
<br />
[2] Stephen Y. K. Seah, et al., '''Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls''', ''Journal of Bacteriology'' 183:1511–1516 (2001)<br />
<br />
[3] Sang-ho Park, et al., '''Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants''', ''Environmental Monitoring and Assessment'' 92:241–251 (2004)<br />
<br />
[4] Qinhong Wang, et al., '''Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery''', ''Biotechnology and Bioengineering'', 98:842-853 (2007)<br />
<br />
[5] Jessika Feliciano, et al., '''ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls''', ''Analytical and Bioanalytical Chemistry'', 385: 807–813 (2006)<br />
<br />
== Results ==<br />
===1. Protocols===<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
===2. Plasmids constructed===<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
===3. A functional part ===<br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector.<br />
<br />
== Acknowledgments ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* Professor David N. Dowling Department of Microbiology, University College, Cork, Ireland<br />
<br />
For the bacteria E. coil SMl0.<br />
<br />
* Professor Junfeng Niu, School of Enviroment, Beijing Normal University <br />
<br />
For his instructions and advices<br />
<br />
* [[Team:Tsinghua|Tsinghua Team]]<br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* [[Team:Chiba|Chiba Team]]<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* [[Team:Tokyo_Tech|Tokyo Tech Team]]<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* [[Team:USTC|USTC Team]]<br />
<br />
For suggestions on our experiments and delicious food in Hefei.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T03:38:29Z<p>Sally730: /* Acknowledgments */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', 105:433–449 (2008), permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== Design Abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal.<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
=== References for special protocol: ===<br />
1. David E. Birch et al., '''Simplified hot start PCR''', ''Nature'' 381:445-446 (1996)<br />
<br />
2. S.Kaijalainen et al., '''An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose''', ''Nucleic Acids Research'' 21:2959-2960 (1993)<br />
<br />
3. Yukihiro Kitade et al., '''Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta) gene''', ''Journal of Applied Phycology'' 15:555-557 (2003)<br />
<br />
4. Markus Ralser et al., '''An efficient and economic enhancer mix for PCR''', ''Biochemical and Biophysical Research Communications'' 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
===1. Switch on the degradation pathway===<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
===2. Solve the bottleneck in PCBs degradation pathway===<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
===3. Control on the amount of PCBs molecular that enter the cell.===<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
===4. System guarantee===<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
===References for Design Details:===<br />
<br />
[1] Kensuke Furukawa, et al., '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of Bioscience and Bioengineering'' 105:433–449 (2008)<br />
<br />
[2] Stephen Y. K. Seah, et al., '''Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls''', ''Journal of Bacteriology'' 183:1511–1516 (2001)<br />
<br />
[3] Sang-ho Park, et al., '''Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants''', ''Environmental Monitoring and Assessment'' 92:241–251 (2004)<br />
<br />
[4] Qinhong Wang, et al., '''Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery''', ''Biotechnology and Bioengineering'', 98:842-853 (2007)<br />
<br />
[5] Jessika Feliciano, et al., '''ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls''', ''Analytical and Bioanalytical Chemistry'', 385: 807–813 (2006)<br />
<br />
== Results ==<br />
===1. Protocols===<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
===2. Plasmids constructed===<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
===3. A functional part ===<br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector.<br />
<br />
== Acknowledgments ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* David N. Dowling<br />
<br />
For the bacteria E. coil SMl0.<br />
<br />
* Professor Niu Junfeng in School of Enviroment, Beijing Normal University <br />
<br />
For his instructions and advices<br />
<br />
* Tsinghua Team <br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* Chiba Team<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* Tokyo Tech Team<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* USTC Team<br />
<br />
For suggestions on our experiments and delicious food in Hefei.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T00:50:01Z<p>Sally730: /* Acknowledgement */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', Vol. 105, No. 5, 433–449. 2008, permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== Design Abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal.<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
<br />
<br />
== References for special protocol: ==<br />
1.Simplified hot start PCR, David E. Birch et al. Nature 381:445-446 (1996)<br />
<br />
2.An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose, S.Kaijalainen et al. Nucleic Acids Research 21:2959-2960 (1993)<br />
<br />
3.Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta)gene, Yukihiro Kitade et al. Journal of Applied Phycology 15:555-557 (2003)<br />
<br />
4.An efficient and economic enhancer mix for PCR, Markus Ralser et al. Biochemical and Biophysical Research Communications 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
Design details<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
1. Switch on the degradation pathway<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
2. Solve the bottleneck in PCBs degradation pathway<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
3. Control on the amount of PCBs molecular that enter the cell.<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
4. System guarantee<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
Reference: <br />
<br />
[1] Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features. Kensuke Furukawa and Hidehiko Fujihara, Journal of bioscience and bioengineering, 2008, Vol. 105, No. 5:433–449,<br />
<br />
[2] Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls. Seah S. Y., Labbe, G., Kaschabek, S. R., Reifenrath, F.,<br />
Reineke, F., and Eltis, L. D., J. Bacteriol., 2001, 183:1511–1516.<br />
<br />
[3] Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants. Sang-ho Park,Young Lee, Jong-Chan Chae and Chi-Kyung Kim. Environmental Monitoring and Assessment, 2004, 92:241–251.<br />
<br />
[4] Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery. Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard, Yongchun Tang. Biotechnology and Bioengineering, 2007, Vol. 98, No. 4.<br />
<br />
[5] ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls. Jessika Feliciano, Shifen Xu, Xiyuan Guan, Hans-Joachim Lehmle , Leonidas G. Bachas, Sylvia Daunert. Anal Bioanal Chem, 2006, 385: 807–813<br />
<br />
== Results ==<br />
1. Protocols<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
2. Plasmids constructed<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
3. A functional part <br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector. <br />
<br />
<br />
== Acknowledgement ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* David N. Dowling<br />
<br />
For the bacteria E. coil SMl0.<br />
<br />
* Professor Niu Junfeng<br />
<br />
For his instructions and advices<br />
<br />
* Tsinghua Team <br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* Chiba Team<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* Tokyo Tech Team<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* USTC Team<br />
<br />
For suggestions on our experiments and delicious food in Hefei.<br />
<br />
* All the BNU menbers<br />
<br />
Of course, we encountered many obstacles and difficulties such as financial problems, cooperation and communication problems of teammates, spirit pressure, time management and so on. But all the hardship turned out to be a precious experience that helped us grow up. Anyway, iGem is one of our most precious experiences and we will never forget these days and night in the lab and meeting room, all the laughs and tears. We have learned a lot in this project except attending the competition itself. <br />
<br />
Nothing can be achieved without all the members' hard work and contribution. Thank you, all the BNU_Team menbers.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T00:48:29Z<p>Sally730: /* Acknowledgement */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', Vol. 105, No. 5, 433–449. 2008, permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== Design Abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal.<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
<br />
<br />
== References for special protocol: ==<br />
1.Simplified hot start PCR, David E. Birch et al. Nature 381:445-446 (1996)<br />
<br />
2.An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose, S.Kaijalainen et al. Nucleic Acids Research 21:2959-2960 (1993)<br />
<br />
3.Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta)gene, Yukihiro Kitade et al. Journal of Applied Phycology 15:555-557 (2003)<br />
<br />
4.An efficient and economic enhancer mix for PCR, Markus Ralser et al. Biochemical and Biophysical Research Communications 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
Design details<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
1. Switch on the degradation pathway<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
2. Solve the bottleneck in PCBs degradation pathway<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
3. Control on the amount of PCBs molecular that enter the cell.<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
4. System guarantee<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
Reference: <br />
<br />
[1] Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features. Kensuke Furukawa and Hidehiko Fujihara, Journal of bioscience and bioengineering, 2008, Vol. 105, No. 5:433–449,<br />
<br />
[2] Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls. Seah S. Y., Labbe, G., Kaschabek, S. R., Reifenrath, F.,<br />
Reineke, F., and Eltis, L. D., J. Bacteriol., 2001, 183:1511–1516.<br />
<br />
[3] Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants. Sang-ho Park,Young Lee, Jong-Chan Chae and Chi-Kyung Kim. Environmental Monitoring and Assessment, 2004, 92:241–251.<br />
<br />
[4] Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery. Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard, Yongchun Tang. Biotechnology and Bioengineering, 2007, Vol. 98, No. 4.<br />
<br />
[5] ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls. Jessika Feliciano, Shifen Xu, Xiyuan Guan, Hans-Joachim Lehmle , Leonidas G. Bachas, Sylvia Daunert. Anal Bioanal Chem, 2006, 385: 807–813<br />
<br />
== Results ==<br />
1. Protocols<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
2. Plasmids constructed<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
3. A functional part <br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector. <br />
<br />
<br />
== Acknowledgement ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* David N. Dowling<br />
<br />
For the bacteria E. coil SMl0.<br />
<br />
* Professor Niu Junfeng<br />
<br />
For his instructions and advices<br />
<br />
* Tsinghua Team <br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* Chiba Team<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* Tokyo Tech Team<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* USTC Team<br />
<br />
For suggestions on our experiments and delicious food in Hefei.<br />
<br />
* All the BNU menbers<br />
<br />
Of course, we encountered many obstacles and difficulties such as financial problems, cooperation and communication problems of teammates, spirit pressure, time management and so on. But all the hardship turned out to be a precious experience that helped us grow up. Anyway, iGem is one of our most precious experiences and we will never forget these days and night in the lab and meeting room, all the laughs and tears. We have learned a lot in this project except the attending the competition itself. <br />
<br />
Nothing can be achieved without all the members' hard work and contribution. Thank you, all the BNU_Team menbers.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/NotebookTeam:Beijing Normal/Notebook2008-10-30T00:45:10Z<p>Sally730: /* Dynamics */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
=='''Dynamics'''==<br />
*== '''Communications''' ==<br />
**17th June: Attend the Asian Workshop meeting.<br />
<br />
**17th July: Meet up with Tsinghua Team, and learn fron their experiences.<br />
<br />
**25th July: With Invitrogen.Co. Ltd and Tsinghua team to sign up a contract.<br />
<br />
**27th July: Have a discussion with Tsinghua team about the transformation efficiency. <br />
We generalize some key words from the discussion: <br />
<br />
1. Make sure the concentration of the plasmids from the biobrick is enough for transformation. <br />
<br />
2. Keep a low temprature when enzyme digestion is performed <br />
<br />
3. The transformation product are always incubated for more than 1h. <br />
<br />
<br />
**9th September: We and Tsinghua team get together in our lab. We have a happy time together and share a lot. <br />
<br />
**10th September: We provide Tsinghua Team with the cells harboring pSB1AC3 plasmids. We did the the tramsformation for them. Because they found their transformation effeciency is relatively low.<br />
<br />
**17th September: Tsinghua Team told us that the vector that we provided proved to work well. We were all pretty happy about this positive result.<br />
<br />
**3th October: We and Chiba Team had a happy conversation via googletalk today. We share all the success and frustrations in the process. And we exchange our opinion about promotor function measurement. They told us that they have used BBa_T9002 because their project uses quorum sensing and the part(T9002) was GFP producer controlled by 3OC6HSL Receiver Device.And their result is pretty satisfying. <br />
<br />
**5th October: We share the experience of measurment with Tokyo Tech University Team. They tell us to use BBa_I13522 (http://partsregistry.org/Part:BBa_I13522) as a positive control when we measure the function of promotors.<br />
<br />
**9th October: We test the founction of Part BBa_I719015. The result indicates that it works well. The cells (BL21(DE3) as host) turn green when induced by IPTG.<br />
<br />
**15th October: Our T-shirt is designed today. It is very pretty. See image below.<br />
<br />
**19th, 20th October: We have a good discussion with USTC team in Hefei, and they give us several important suggestions on experiments.<br />
<br />
[[Image:t-shirt.jpg]]<br />
<br />
[[Image:t-shirt1.jpg]]<br />
<br />
'''Our logo is designed finally. It is a visual field through a microscope. You could see us in this microscope.'''<br />
<br />
[[Image:our logo.jpg]]<br />
<br />
** Our iGem is coming to the end. But our projet will continue. <br />
<br />
Of course, we encountered many obstacles and difficulties such as financial problems, cooperation and communication problems of teammates, spirit pressure, time management and so on. But all the hardship turned out to be a precious experience that helped us grow up. Anyway, iGem is one of our most precious experiences and we will never forget these days and night in the lab and meeting room, all the laughs and tears. We have learned a lot in this project except the attending the competition itself. <br />
<br />
<br />
<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=07}}<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=08}}<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=09}}<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=10}}<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=11}}</div>Sally730http://2008.igem.org/Team:Beijing_Normal/NotebookTeam:Beijing Normal/Notebook2008-10-30T00:38:36Z<p>Sally730: /* Dynamics */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
=='''Dynamics'''==<br />
*== '''Communications''' ==<br />
**17th June: Attend the Asian Workshop meeting.<br />
<br />
**17th July: Meet up with Tsinghua Team, and learn fron their experiences.<br />
<br />
**25th July: With Invitrogen.Co. Ltd and Tsinghua team to sign up a contract.<br />
<br />
**27th July: Have a discussion with Tsinghua team about the transformation efficiency. <br />
We generalize some key words from the discussion: <br />
<br />
1. Make sure the concentration of the plasmids from the biobrick is enough for transformation. <br />
<br />
2. Keep a low temprature when enzyme digestion is performed <br />
<br />
3. The transformation product are always incubated for more than 1h. <br />
<br />
<br />
**9th September: We and Tsinghua team get together in our lab. We have a happy time together and share a lot. <br />
<br />
**10th September: We provide Tsinghua Team with the cells harboring pSB1AC3 plasmids. We did the the tramsformation for them. Because they found their transformation effeciency is relatively low.<br />
<br />
**17th September: Tsinghua Team told us that the vector that we provided proved to work well. We were all pretty happy about this positive result.<br />
<br />
**3th October: We and Chiba Team had a happy conversation via googletalk today. We share all the success and frustrations in the process. And we exchange our opinion about promotor function measurement. They told us that they have used BBa_T9002 because their project uses quorum sensing and the part(T9002) was GFP producer controlled by 3OC6HSL Receiver Device.And their result is pretty satisfying. <br />
<br />
**5th October: We share the experience of measurment with Tokyo Tech University Team. They tell us to use BBa_I13522 (http://partsregistry.org/Part:BBa_I13522) as a positive control when we measure the function of promotors.<br />
<br />
**9th October: We test the founction of Part BBa_I719015. The result indicates that it works well. The cells (BL21(DE3) as host) turn green when induced by IPTG.<br />
<br />
**15th October: Our T-shirt is designed today. It is very pretty. See image below.<br />
<br />
**19th, 20th October: We have a good discussion with USTC team in Hefei, and they give us several important suggestions on experiments.<br />
<br />
[[Image:t-shirt.jpg]]<br />
<br />
[[Image:t-shirt1.jpg]]<br />
<br />
'''Our logo is designed finally. It is a visual field through a microscope. You could see us in this microscope.'''<br />
<br />
[[Image:our logo.jpg]]<br />
<br />
** Our iGem is coming to the end. But our projet will continue. We have a lot of fun in this process. <br />
<br />
<br />
<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=07}}<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=08}}<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=09}}<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=10}}<br />
{{ #calendar: title=Beijing_Normal |year=2008|month=11}}</div>Sally730http://2008.igem.org/Team:Beijing_Normal/TeamTeam:Beijing Normal/Team2008-10-30T00:29:05Z<p>Sally730: /* Who we are */</p>
<hr />
<div>__NOTOC__<br />
{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== This is Team Beijing Normal ==<br />
<br />
[[Image:Example_bnu logo.png]] <br />
<br />
The logo above is ours. The idea behind the design is that you can see us from a microscope, and it has been magnified 400 times. The person in the middle of the cell is <b>i</b>, one of us. The bulb is our idea, inside a cell.<br />
<br />
[[Image:team bnu.jpg|thumb|720px|center|Team members in China. From left: Ya, Jingyi, Sheng, Gang]]<br />
<br />
==Our lab==<br />
<gallery><br />
Image: BNUlab001.jpg<br />
Image: BNUlab002.jpg<br />
Image: BNUlab003.jpg<br />
Image: BNUlab004.jpg<br />
Image: BNUlab005.jpg<br />
Image: BNUlab006.jpg<br />
Image: BNUlab007.jpg<br />
Image: BNUlab008.jpg<br />
</gallery><br />
<br />
== Who we are ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
<br />
'''Advisors:'''<br />
<br />
*''' Advisor 1''': Junfeng Niu<br />
*'''Advisor 2''': Shi Chen<br />
<br />
'''Students:'''<br />
<br />
*'''Student 1''': [[User:proline|Gang Wu]]<br />
*'''Student 2''': [[User:Sally730|Jingyi Wang]]<br />
*'''Student 3''': [[User:wonderland|Ya He]]<br />
*'''Student 4''': [[User:viva8565 | Sheng Feng]]<br />
*'''Student 5''': [[User:Forrest_Bao|Forrest Sheng Bao]]<br />
<br />
|<br />
<gallery><br />
Image:Forrest.JPG| [[User:Forrest_Bao|Forrest Sheng Bao]]<br />
Image:Sally.jpg|[[User:Sally730|Jingyi Wang]]<br />
Image:Jenny.jpg|[[User:wonderland|Ya He]]<br />
Image:Team_member_4.png|[[User:proline|Gang Wu]]<br />
Image:Team_member_5.png|[[User:viva8565 | Sheng Feng]]<br />
Image:Lavi.jpg|Shi Chen<br />
</gallery><br />
|}<br />
<br />
== Where we are from ==<br />
[[User:Forrest_Bao|Forrest Sheng Bao]] is from Dept. of Computer Science and Dept. of Electrical Engineering, Texas Tech University, Texas. He is a master student in Electrical Engineering and PhD candidate in Computer Science. He obtained a bachelor degree in electrical engineering in 2006.<br />
<br />
[[User:Sally730|Jingyi Wang]] is a senior of environmental science and technology, Beijing Normal University.<br />
<br />
[[User:wonderland|Ya He]] is a fourth year student studying Environmental engineering at Beijing Normal University. She had previously spent most of her childhood in Oxford, UK.<br />
<br />
[[User:proline | Gang Wu]] graduated from Department of biochemsitry, Nanjing University in 2006.<br />
<br />
[[User:viva8565 | Sheng Feng]] is a master student from College of Life science, Beijing Normal University.<br />
<br />
Shi Chen is Ph.D candidate of Entomology at Penn. State Univ. Currently he is also a Ph.D dual-degree student majoring Operations Research. His research interests are broad and include animal predation behavior, insect life history analysis, integrated pest management, insect wing venation analysis and water pollution prediction, etc. He likes to use mathematical, statistical and computational techniques to solve real world problems. His extracurricular interests include classical literature, music and photography. He really cherishes this chance of iGEM that I could work with many friends and have great communication with them.<br />
<br />
== What we have done ==<br />
===Forrest===<br />
[[User:Forrest_Bao|Forrest Sheng Bao]]'s research interests are in artificial intelligence, biomedical engineering and bioinformatics. He is very interested on algorithms in biology and medicine fields, as well as the interdisciplinary fields of computing and biology.<br />
<br />
This is his<br />
<html><br />
publications:</span><br />
<div style="margin-left: 10px;"><br />
<ul><br />
<li><u>Forrest Sheng Bao</u>, <i>et al.</i>, Automated Recognition and Diagnosis of Epilepsy Using EEG and a Probabilistic Neural Network, <i>20th IEEE Int'l Conference on Tools with Artificial Intelligence (ICTAI 2008)</i>, Nov. 2008, To appear</li><br />
<li>Lei Yuan and <u>Forrest Sheng Bao</u>, Automatic Analysis of Facial Expressions of Acute Pain in Neonates Using Boosted Gabor Features, <i>20th IEEE Int'l Conference on Tools with Artificial Intelligence (ICTAI 2008)</i>, Nov. 2008, To appear<br />
<li>Yijin Zhang, <u>Forrest Sheng Bao</u>, <i>et al</i>., Analysis of Energy Efficiency and Power Saving in IEEE 802.15.4, <i>IEEE WCNC 2007</i>, 2007</li><br />
<li>Yu-Xuan Wang and <u>Forrest Sheng Bao</u>, An Entropy-based Weighted Clustering Algorithm and Its Optimization for Ad hoc Networks, <i>IEEE WiMob 2007</i>, 2007</li><br />
<li>Stephen Gang Wu, <u>Forrest Sheng Bao</u>, Eric You Xu, Yu-Xuan Wang, Yi-Fan Chang and Chiao-Liang Shiang, A Leaf Recognition Algorithm for Plant classification Using Probabilistic Neural Network, <i>IEEE ISSPIT 2007</i>, 2007</li><br />
<li>Zhendong Zhao, Lei Yuan, Yuxuan Wang, <u>Forrest Sheng Bao</u>,A Novel Model of Working Set Selection for SMO Decomposition Methods, <i>IEEE ICTAI 2007</i>, 2007</li><br />
<br />
</ul><br />
</div><br />
</html><br />
<br />
===Jingyi===<br />
<br />
As a new graduate, [[User:Sally730 | Jingyi Wang]] does not have much experience in research and the program. From Nov. 2007 till now, she is working on the project "Detection of chlorophenols in aqueous phase using an Enzyme Thermistor", which is a China national 863 program. She does research on a laccase enzyme column based biosensor using thermometric measurement to detect chlorophenals pollutants. From May 2007 to Dec. 2007, she was involved in the project "Analysis of the distribution of PAHs in the conservation zone of Huang He estuary", which is a China national 973 project. Her job in the project is sampling, extracting, detecting and analysis PAHs in aqueous phase and solid phase, as well as assessing the ecological risk of the region.<br />
<br />
===Gang===<br />
<br />
[[User:proline|Gang Wu]] holds a China software patent "An Automatic System of Leaf Recognition" (NO.2006118213) and has one publication "A Leaf Recognition Algorithm for Plant Classification Using Probabilistic Neural Network", IEEE International Symposium on Signal Processing and Information Technology 2007, p12 - 17, 2007.<br />
<br />
From Mar. 2007 till now, he is working on the project "Self-regulatory gene network and new components for gene regulation", His job is to implement the classical Lac regulatory system to design a self-repression genetic switch. Learn the properties of sRNA in regulatory circuit. During Aug.2005 to Sept.2005 and Mar.2007, he utilized an SIRS model to analyze the pollution and decontamination information of past ten years and predict the tendency and quality of water in next twenty years in the project "A water pollution and decontamination model for Yangtze River". From Mar. 2005 to Jun. 2005, he was involved in the project "Microbial degradation of PU (polyurethane)". He collected and selected PU degradation microbes from environment using classical method of microbiology, participator.<br />
<br />
===Ya===<br />
Ya's previous research experience includes chemical engineering and supply chain optimization. She participated in the Hi-tech Research and Development Program of China (No. 2006AA06Z323): Investigating New Methods Using Laccase as Photocatalyst to Degrade Chlorophenols. She was mainly investigating photocatalysis and the synergistic effect of different catalysis reacting in one system. She has also been engaged in research adopting the system approach to achieve the most optimum and sustainable utility of supply chain. <br />
<br />
===Sheng===<br />
From Sept. 2007 till now, [[User:viva8565| Sheng]] is working on the project "Proteomic Study on Progression and Metastasis of Cancer", directed by Prof. Xueyuan Xiao (Universitis’ Conferderated Institute of Proteomics, Beijing Normal University) to study the tumor bio-markers and their function in progression and metastasis of cancer through proteomics method.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/TeamTeam:Beijing Normal/Team2008-10-30T00:28:37Z<p>Sally730: /* Who we are */</p>
<hr />
<div>__NOTOC__<br />
{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== This is Team Beijing Normal ==<br />
<br />
[[Image:Example_bnu logo.png]] <br />
<br />
The logo above is ours. The idea behind the design is that you can see us from a microscope, and it has been magnified 400 times. The person in the middle of the cell is <b>i</b>, one of us. The bulb is our idea, inside a cell.<br />
<br />
[[Image:team bnu.jpg|thumb|720px|center|Team members in China. From left: Ya, Jingyi, Sheng, Gang]]<br />
<br />
==Our lab==<br />
<gallery><br />
Image: BNUlab001.jpg<br />
Image: BNUlab002.jpg<br />
Image: BNUlab003.jpg<br />
Image: BNUlab004.jpg<br />
Image: BNUlab005.jpg<br />
Image: BNUlab006.jpg<br />
Image: BNUlab007.jpg<br />
Image: BNUlab008.jpg<br />
</gallery><br />
<br />
== Who we are ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
<br />
'''Advisors:'''<br />
<br />
*''' Advisor 1''': Junfeng Niu<br />
*'''Advisor 2''': Shi Chen<br />
<br />
'''Students:'''<br />
<br />
*'''Student 1''': [[User:proline|Gang Wu]]<br />
*'''Student 2''': [[User:Sally730|Jingyi Wang]]<br />
*'''Student 3''': [[User:wonderland|Ya He]]<br />
*'''Student 4''': [[User:viva8565 | Sheng Feng]]<br />
*'''Student 5''': [[User:Forrest_Bao|Forrest Sheng Bao]]<br />
<br />
|<br />
<gallery><br />
Image:Forrest.JPG| [[User:Forrest_Bao|Forrest Sheng Bao]]<br />
Image:Sally.jpg|[[User:Sally730|Jingyi Wang]]<br />
Image:Jenny.jpg|[[User:wonderland|Ya He]]<br />
Image:Team_member_4.png|[[User:proline|Gang Wu]]<br />
Image:Team_member_5.jpg|[[User:viva8565 | Sheng Feng]]<br />
Image:Lavi.jpg|Shi Chen<br />
</gallery><br />
|}<br />
<br />
== Where we are from ==<br />
[[User:Forrest_Bao|Forrest Sheng Bao]] is from Dept. of Computer Science and Dept. of Electrical Engineering, Texas Tech University, Texas. He is a master student in Electrical Engineering and PhD candidate in Computer Science. He obtained a bachelor degree in electrical engineering in 2006.<br />
<br />
[[User:Sally730|Jingyi Wang]] is a senior of environmental science and technology, Beijing Normal University.<br />
<br />
[[User:wonderland|Ya He]] is a fourth year student studying Environmental engineering at Beijing Normal University. She had previously spent most of her childhood in Oxford, UK.<br />
<br />
[[User:proline | Gang Wu]] graduated from Department of biochemsitry, Nanjing University in 2006.<br />
<br />
[[User:viva8565 | Sheng Feng]] is a master student from College of Life science, Beijing Normal University.<br />
<br />
Shi Chen is Ph.D candidate of Entomology at Penn. State Univ. Currently he is also a Ph.D dual-degree student majoring Operations Research. His research interests are broad and include animal predation behavior, insect life history analysis, integrated pest management, insect wing venation analysis and water pollution prediction, etc. He likes to use mathematical, statistical and computational techniques to solve real world problems. His extracurricular interests include classical literature, music and photography. He really cherishes this chance of iGEM that I could work with many friends and have great communication with them.<br />
<br />
== What we have done ==<br />
===Forrest===<br />
[[User:Forrest_Bao|Forrest Sheng Bao]]'s research interests are in artificial intelligence, biomedical engineering and bioinformatics. He is very interested on algorithms in biology and medicine fields, as well as the interdisciplinary fields of computing and biology.<br />
<br />
This is his<br />
<html><br />
publications:</span><br />
<div style="margin-left: 10px;"><br />
<ul><br />
<li><u>Forrest Sheng Bao</u>, <i>et al.</i>, Automated Recognition and Diagnosis of Epilepsy Using EEG and a Probabilistic Neural Network, <i>20th IEEE Int'l Conference on Tools with Artificial Intelligence (ICTAI 2008)</i>, Nov. 2008, To appear</li><br />
<li>Lei Yuan and <u>Forrest Sheng Bao</u>, Automatic Analysis of Facial Expressions of Acute Pain in Neonates Using Boosted Gabor Features, <i>20th IEEE Int'l Conference on Tools with Artificial Intelligence (ICTAI 2008)</i>, Nov. 2008, To appear<br />
<li>Yijin Zhang, <u>Forrest Sheng Bao</u>, <i>et al</i>., Analysis of Energy Efficiency and Power Saving in IEEE 802.15.4, <i>IEEE WCNC 2007</i>, 2007</li><br />
<li>Yu-Xuan Wang and <u>Forrest Sheng Bao</u>, An Entropy-based Weighted Clustering Algorithm and Its Optimization for Ad hoc Networks, <i>IEEE WiMob 2007</i>, 2007</li><br />
<li>Stephen Gang Wu, <u>Forrest Sheng Bao</u>, Eric You Xu, Yu-Xuan Wang, Yi-Fan Chang and Chiao-Liang Shiang, A Leaf Recognition Algorithm for Plant classification Using Probabilistic Neural Network, <i>IEEE ISSPIT 2007</i>, 2007</li><br />
<li>Zhendong Zhao, Lei Yuan, Yuxuan Wang, <u>Forrest Sheng Bao</u>,A Novel Model of Working Set Selection for SMO Decomposition Methods, <i>IEEE ICTAI 2007</i>, 2007</li><br />
<br />
</ul><br />
</div><br />
</html><br />
<br />
===Jingyi===<br />
<br />
As a new graduate, [[User:Sally730 | Jingyi Wang]] does not have much experience in research and the program. From Nov. 2007 till now, she is working on the project "Detection of chlorophenols in aqueous phase using an Enzyme Thermistor", which is a China national 863 program. She does research on a laccase enzyme column based biosensor using thermometric measurement to detect chlorophenals pollutants. From May 2007 to Dec. 2007, she was involved in the project "Analysis of the distribution of PAHs in the conservation zone of Huang He estuary", which is a China national 973 project. Her job in the project is sampling, extracting, detecting and analysis PAHs in aqueous phase and solid phase, as well as assessing the ecological risk of the region.<br />
<br />
===Gang===<br />
<br />
[[User:proline|Gang Wu]] holds a China software patent "An Automatic System of Leaf Recognition" (NO.2006118213) and has one publication "A Leaf Recognition Algorithm for Plant Classification Using Probabilistic Neural Network", IEEE International Symposium on Signal Processing and Information Technology 2007, p12 - 17, 2007.<br />
<br />
From Mar. 2007 till now, he is working on the project "Self-regulatory gene network and new components for gene regulation", His job is to implement the classical Lac regulatory system to design a self-repression genetic switch. Learn the properties of sRNA in regulatory circuit. During Aug.2005 to Sept.2005 and Mar.2007, he utilized an SIRS model to analyze the pollution and decontamination information of past ten years and predict the tendency and quality of water in next twenty years in the project "A water pollution and decontamination model for Yangtze River". From Mar. 2005 to Jun. 2005, he was involved in the project "Microbial degradation of PU (polyurethane)". He collected and selected PU degradation microbes from environment using classical method of microbiology, participator.<br />
<br />
===Ya===<br />
Ya's previous research experience includes chemical engineering and supply chain optimization. She participated in the Hi-tech Research and Development Program of China (No. 2006AA06Z323): Investigating New Methods Using Laccase as Photocatalyst to Degrade Chlorophenols. She was mainly investigating photocatalysis and the synergistic effect of different catalysis reacting in one system. She has also been engaged in research adopting the system approach to achieve the most optimum and sustainable utility of supply chain. <br />
<br />
===Sheng===<br />
From Sept. 2007 till now, [[User:viva8565| Sheng]] is working on the project "Proteomic Study on Progression and Metastasis of Cancer", directed by Prof. Xueyuan Xiao (Universitis’ Conferderated Institute of Proteomics, Beijing Normal University) to study the tumor bio-markers and their function in progression and metastasis of cancer through proteomics method.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/TeamTeam:Beijing Normal/Team2008-10-30T00:26:53Z<p>Sally730: /* Who we are */</p>
<hr />
<div>__NOTOC__<br />
{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== This is Team Beijing Normal ==<br />
<br />
[[Image:Example_bnu logo.png]] <br />
<br />
The logo above is ours. The idea behind the design is that you can see us from a microscope, and it has been magnified 400 times. The person in the middle of the cell is <b>i</b>, one of us. The bulb is our idea, inside a cell.<br />
<br />
[[Image:team bnu.jpg|thumb|720px|center|Team members in China. From left: Ya, Jingyi, Sheng, Gang]]<br />
<br />
==Our lab==<br />
<gallery><br />
Image: BNUlab001.jpg<br />
Image: BNUlab002.jpg<br />
Image: BNUlab003.jpg<br />
Image: BNUlab004.jpg<br />
Image: BNUlab005.jpg<br />
Image: BNUlab006.jpg<br />
Image: BNUlab007.jpg<br />
Image: BNUlab008.jpg<br />
</gallery><br />
<br />
== Who we are ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
<br />
'''Advisors:'''<br />
<br />
*''' Advisor 1''': Junfeng Niu<br />
*'''Advisor 2''': Shi Chen<br />
<br />
'''Students:'''<br />
<br />
*'''Student 1''': [[User:proline|Gang Wu]]<br />
*'''Student 2''': [[User:Sally730|Jingyi Wang]]<br />
*'''Student 3''': [[User:wonderland|Ya He]]<br />
*'''Student 4''': [[User:viva8565 | Sheng Feng]]<br />
*'''Student 5''': [[User:Forrest_Bao|Forrest Sheng Bao]]<br />
<br />
|<br />
<gallery><br />
Image:Forrest.JPG| [[User:Forrest_Bao|Forrest Sheng Bao]]<br />
Image:Sally.jpg|[[User:Sally730|Jingyi Wang]]<br />
Image:Jenny.jpg|[[User:wonderland|Ya He]]<br />
Image:Team_member_4.png|[[User:proline|Gang Wu]]<br />
Image:fengsheng.jpg|[[User:viva8565 | Sheng Feng]]<br />
Image:Lavi.jpg|Shi Chen<br />
</gallery><br />
|}<br />
<br />
== Where we are from ==<br />
[[User:Forrest_Bao|Forrest Sheng Bao]] is from Dept. of Computer Science and Dept. of Electrical Engineering, Texas Tech University, Texas. He is a master student in Electrical Engineering and PhD candidate in Computer Science. He obtained a bachelor degree in electrical engineering in 2006.<br />
<br />
[[User:Sally730|Jingyi Wang]] is a senior of environmental science and technology, Beijing Normal University.<br />
<br />
[[User:wonderland|Ya He]] is a fourth year student studying Environmental engineering at Beijing Normal University. She had previously spent most of her childhood in Oxford, UK.<br />
<br />
[[User:proline | Gang Wu]] graduated from Department of biochemsitry, Nanjing University in 2006.<br />
<br />
[[User:viva8565 | Sheng Feng]] is a master student from College of Life science, Beijing Normal University.<br />
<br />
Shi Chen is Ph.D candidate of Entomology at Penn. State Univ. Currently he is also a Ph.D dual-degree student majoring Operations Research. His research interests are broad and include animal predation behavior, insect life history analysis, integrated pest management, insect wing venation analysis and water pollution prediction, etc. He likes to use mathematical, statistical and computational techniques to solve real world problems. His extracurricular interests include classical literature, music and photography. He really cherishes this chance of iGEM that I could work with many friends and have great communication with them.<br />
<br />
== What we have done ==<br />
===Forrest===<br />
[[User:Forrest_Bao|Forrest Sheng Bao]]'s research interests are in artificial intelligence, biomedical engineering and bioinformatics. He is very interested on algorithms in biology and medicine fields, as well as the interdisciplinary fields of computing and biology.<br />
<br />
This is his<br />
<html><br />
publications:</span><br />
<div style="margin-left: 10px;"><br />
<ul><br />
<li><u>Forrest Sheng Bao</u>, <i>et al.</i>, Automated Recognition and Diagnosis of Epilepsy Using EEG and a Probabilistic Neural Network, <i>20th IEEE Int'l Conference on Tools with Artificial Intelligence (ICTAI 2008)</i>, Nov. 2008, To appear</li><br />
<li>Lei Yuan and <u>Forrest Sheng Bao</u>, Automatic Analysis of Facial Expressions of Acute Pain in Neonates Using Boosted Gabor Features, <i>20th IEEE Int'l Conference on Tools with Artificial Intelligence (ICTAI 2008)</i>, Nov. 2008, To appear<br />
<li>Yijin Zhang, <u>Forrest Sheng Bao</u>, <i>et al</i>., Analysis of Energy Efficiency and Power Saving in IEEE 802.15.4, <i>IEEE WCNC 2007</i>, 2007</li><br />
<li>Yu-Xuan Wang and <u>Forrest Sheng Bao</u>, An Entropy-based Weighted Clustering Algorithm and Its Optimization for Ad hoc Networks, <i>IEEE WiMob 2007</i>, 2007</li><br />
<li>Stephen Gang Wu, <u>Forrest Sheng Bao</u>, Eric You Xu, Yu-Xuan Wang, Yi-Fan Chang and Chiao-Liang Shiang, A Leaf Recognition Algorithm for Plant classification Using Probabilistic Neural Network, <i>IEEE ISSPIT 2007</i>, 2007</li><br />
<li>Zhendong Zhao, Lei Yuan, Yuxuan Wang, <u>Forrest Sheng Bao</u>,A Novel Model of Working Set Selection for SMO Decomposition Methods, <i>IEEE ICTAI 2007</i>, 2007</li><br />
<br />
</ul><br />
</div><br />
</html><br />
<br />
===Jingyi===<br />
<br />
As a new graduate, [[User:Sally730 | Jingyi Wang]] does not have much experience in research and the program. From Nov. 2007 till now, she is working on the project "Detection of chlorophenols in aqueous phase using an Enzyme Thermistor", which is a China national 863 program. She does research on a laccase enzyme column based biosensor using thermometric measurement to detect chlorophenals pollutants. From May 2007 to Dec. 2007, she was involved in the project "Analysis of the distribution of PAHs in the conservation zone of Huang He estuary", which is a China national 973 project. Her job in the project is sampling, extracting, detecting and analysis PAHs in aqueous phase and solid phase, as well as assessing the ecological risk of the region.<br />
<br />
===Gang===<br />
<br />
[[User:proline|Gang Wu]] holds a China software patent "An Automatic System of Leaf Recognition" (NO.2006118213) and has one publication "A Leaf Recognition Algorithm for Plant Classification Using Probabilistic Neural Network", IEEE International Symposium on Signal Processing and Information Technology 2007, p12 - 17, 2007.<br />
<br />
From Mar. 2007 till now, he is working on the project "Self-regulatory gene network and new components for gene regulation", His job is to implement the classical Lac regulatory system to design a self-repression genetic switch. Learn the properties of sRNA in regulatory circuit. During Aug.2005 to Sept.2005 and Mar.2007, he utilized an SIRS model to analyze the pollution and decontamination information of past ten years and predict the tendency and quality of water in next twenty years in the project "A water pollution and decontamination model for Yangtze River". From Mar. 2005 to Jun. 2005, he was involved in the project "Microbial degradation of PU (polyurethane)". He collected and selected PU degradation microbes from environment using classical method of microbiology, participator.<br />
<br />
===Ya===<br />
Ya's previous research experience includes chemical engineering and supply chain optimization. She participated in the Hi-tech Research and Development Program of China (No. 2006AA06Z323): Investigating New Methods Using Laccase as Photocatalyst to Degrade Chlorophenols. She was mainly investigating photocatalysis and the synergistic effect of different catalysis reacting in one system. She has also been engaged in research adopting the system approach to achieve the most optimum and sustainable utility of supply chain. <br />
<br />
===Sheng===<br />
From Sept. 2007 till now, [[User:viva8565| Sheng]] is working on the project "Proteomic Study on Progression and Metastasis of Cancer", directed by Prof. Xueyuan Xiao (Universitis’ Conferderated Institute of Proteomics, Beijing Normal University) to study the tumor bio-markers and their function in progression and metastasis of cancer through proteomics method.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/TeamTeam:Beijing Normal/Team2008-10-30T00:25:50Z<p>Sally730: /* Who we are */</p>
<hr />
<div>__NOTOC__<br />
{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== This is Team Beijing Normal ==<br />
<br />
[[Image:Example_bnu logo.png]] <br />
<br />
The logo above is ours. The idea behind the design is that you can see us from a microscope, and it has been magnified 400 times. The person in the middle of the cell is <b>i</b>, one of us. The bulb is our idea, inside a cell.<br />
<br />
[[Image:team bnu.jpg|thumb|720px|center|Team members in China. From left: Ya, Jingyi, Sheng, Gang]]<br />
<br />
==Our lab==<br />
<gallery><br />
Image: BNUlab001.jpg<br />
Image: BNUlab002.jpg<br />
Image: BNUlab003.jpg<br />
Image: BNUlab004.jpg<br />
Image: BNUlab005.jpg<br />
Image: BNUlab006.jpg<br />
Image: BNUlab007.jpg<br />
Image: BNUlab008.jpg<br />
</gallery><br />
<br />
== Who we are ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
<br />
'''Advisors:'''<br />
<br />
*''' Advisor 1''': Junfeng Niu<br />
*'''Advisor 2''': Shi Chen<br />
<br />
'''Students:'''<br />
<br />
*'''Student 1''': [[User:proline|Gang Wu]]<br />
*'''Student 2''': [[User:Sally730|Jingyi Wang]]<br />
*'''Student 3''': [[User:wonderland|Ya He]]<br />
*'''Student 4''': [[User:viva8565 | Sheng Feng]]<br />
*'''Student 5''': [[User:Forrest_Bao|Forrest Sheng Bao]]<br />
<br />
|<br />
<gallery><br />
Image:Forrest.JPG| [[User:Forrest_Bao|Forrest Sheng Bao]]<br />
Image:Sally.jpg|[[User:Sally730|Jingyi Wang]]<br />
Image:Jenny.jpg|[[User:wonderland|Ya He]]<br />
Image:Team_member_4.png|[[User:proline|Gang Wu]]<br />
Image:feng.jpg|[[User:viva8565 | Sheng Feng]]<br />
Image:Lavi.jpg|Shi Chen<br />
</gallery><br />
|}<br />
<br />
== Where we are from ==<br />
[[User:Forrest_Bao|Forrest Sheng Bao]] is from Dept. of Computer Science and Dept. of Electrical Engineering, Texas Tech University, Texas. He is a master student in Electrical Engineering and PhD candidate in Computer Science. He obtained a bachelor degree in electrical engineering in 2006.<br />
<br />
[[User:Sally730|Jingyi Wang]] is a senior of environmental science and technology, Beijing Normal University.<br />
<br />
[[User:wonderland|Ya He]] is a fourth year student studying Environmental engineering at Beijing Normal University. She had previously spent most of her childhood in Oxford, UK.<br />
<br />
[[User:proline | Gang Wu]] graduated from Department of biochemsitry, Nanjing University in 2006.<br />
<br />
[[User:viva8565 | Sheng Feng]] is a master student from College of Life science, Beijing Normal University.<br />
<br />
Shi Chen is Ph.D candidate of Entomology at Penn. State Univ. Currently he is also a Ph.D dual-degree student majoring Operations Research. His research interests are broad and include animal predation behavior, insect life history analysis, integrated pest management, insect wing venation analysis and water pollution prediction, etc. He likes to use mathematical, statistical and computational techniques to solve real world problems. His extracurricular interests include classical literature, music and photography. He really cherishes this chance of iGEM that I could work with many friends and have great communication with them.<br />
<br />
== What we have done ==<br />
===Forrest===<br />
[[User:Forrest_Bao|Forrest Sheng Bao]]'s research interests are in artificial intelligence, biomedical engineering and bioinformatics. He is very interested on algorithms in biology and medicine fields, as well as the interdisciplinary fields of computing and biology.<br />
<br />
This is his<br />
<html><br />
publications:</span><br />
<div style="margin-left: 10px;"><br />
<ul><br />
<li><u>Forrest Sheng Bao</u>, <i>et al.</i>, Automated Recognition and Diagnosis of Epilepsy Using EEG and a Probabilistic Neural Network, <i>20th IEEE Int'l Conference on Tools with Artificial Intelligence (ICTAI 2008)</i>, Nov. 2008, To appear</li><br />
<li>Lei Yuan and <u>Forrest Sheng Bao</u>, Automatic Analysis of Facial Expressions of Acute Pain in Neonates Using Boosted Gabor Features, <i>20th IEEE Int'l Conference on Tools with Artificial Intelligence (ICTAI 2008)</i>, Nov. 2008, To appear<br />
<li>Yijin Zhang, <u>Forrest Sheng Bao</u>, <i>et al</i>., Analysis of Energy Efficiency and Power Saving in IEEE 802.15.4, <i>IEEE WCNC 2007</i>, 2007</li><br />
<li>Yu-Xuan Wang and <u>Forrest Sheng Bao</u>, An Entropy-based Weighted Clustering Algorithm and Its Optimization for Ad hoc Networks, <i>IEEE WiMob 2007</i>, 2007</li><br />
<li>Stephen Gang Wu, <u>Forrest Sheng Bao</u>, Eric You Xu, Yu-Xuan Wang, Yi-Fan Chang and Chiao-Liang Shiang, A Leaf Recognition Algorithm for Plant classification Using Probabilistic Neural Network, <i>IEEE ISSPIT 2007</i>, 2007</li><br />
<li>Zhendong Zhao, Lei Yuan, Yuxuan Wang, <u>Forrest Sheng Bao</u>,A Novel Model of Working Set Selection for SMO Decomposition Methods, <i>IEEE ICTAI 2007</i>, 2007</li><br />
<br />
</ul><br />
</div><br />
</html><br />
<br />
===Jingyi===<br />
<br />
As a new graduate, [[User:Sally730 | Jingyi Wang]] does not have much experience in research and the program. From Nov. 2007 till now, she is working on the project "Detection of chlorophenols in aqueous phase using an Enzyme Thermistor", which is a China national 863 program. She does research on a laccase enzyme column based biosensor using thermometric measurement to detect chlorophenals pollutants. From May 2007 to Dec. 2007, she was involved in the project "Analysis of the distribution of PAHs in the conservation zone of Huang He estuary", which is a China national 973 project. Her job in the project is sampling, extracting, detecting and analysis PAHs in aqueous phase and solid phase, as well as assessing the ecological risk of the region.<br />
<br />
===Gang===<br />
<br />
[[User:proline|Gang Wu]] holds a China software patent "An Automatic System of Leaf Recognition" (NO.2006118213) and has one publication "A Leaf Recognition Algorithm for Plant Classification Using Probabilistic Neural Network", IEEE International Symposium on Signal Processing and Information Technology 2007, p12 - 17, 2007.<br />
<br />
From Mar. 2007 till now, he is working on the project "Self-regulatory gene network and new components for gene regulation", His job is to implement the classical Lac regulatory system to design a self-repression genetic switch. Learn the properties of sRNA in regulatory circuit. During Aug.2005 to Sept.2005 and Mar.2007, he utilized an SIRS model to analyze the pollution and decontamination information of past ten years and predict the tendency and quality of water in next twenty years in the project "A water pollution and decontamination model for Yangtze River". From Mar. 2005 to Jun. 2005, he was involved in the project "Microbial degradation of PU (polyurethane)". He collected and selected PU degradation microbes from environment using classical method of microbiology, participator.<br />
<br />
===Ya===<br />
Ya's previous research experience includes chemical engineering and supply chain optimization. She participated in the Hi-tech Research and Development Program of China (No. 2006AA06Z323): Investigating New Methods Using Laccase as Photocatalyst to Degrade Chlorophenols. She was mainly investigating photocatalysis and the synergistic effect of different catalysis reacting in one system. She has also been engaged in research adopting the system approach to achieve the most optimum and sustainable utility of supply chain. <br />
<br />
===Sheng===<br />
From Sept. 2007 till now, [[User:viva8565| Sheng]] is working on the project "Proteomic Study on Progression and Metastasis of Cancer", directed by Prof. Xueyuan Xiao (Universitis’ Conferderated Institute of Proteomics, Beijing Normal University) to study the tumor bio-markers and their function in progression and metastasis of cancer through proteomics method.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/TeamTeam:Beijing Normal/Team2008-10-30T00:25:26Z<p>Sally730: /* Who we are */</p>
<hr />
<div>__NOTOC__<br />
{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== This is Team Beijing Normal ==<br />
<br />
[[Image:Example_bnu logo.png]] <br />
<br />
The logo above is ours. The idea behind the design is that you can see us from a microscope, and it has been magnified 400 times. The person in the middle of the cell is <b>i</b>, one of us. The bulb is our idea, inside a cell.<br />
<br />
[[Image:team bnu.jpg|thumb|720px|center|Team members in China. From left: Ya, Jingyi, Sheng, Gang]]<br />
<br />
==Our lab==<br />
<gallery><br />
Image: BNUlab001.jpg<br />
Image: BNUlab002.jpg<br />
Image: BNUlab003.jpg<br />
Image: BNUlab004.jpg<br />
Image: BNUlab005.jpg<br />
Image: BNUlab006.jpg<br />
Image: BNUlab007.jpg<br />
Image: BNUlab008.jpg<br />
</gallery><br />
<br />
== Who we are ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
<br />
'''Advisors:'''<br />
<br />
*''' Advisor 1''': Junfeng Niu<br />
*'''Advisor 2''': Shi Chen<br />
<br />
'''Students:'''<br />
<br />
*'''Student 1''': [[User:proline|Gang Wu]]<br />
*'''Student 2''': [[User:Sally730|Jingyi Wang]]<br />
*'''Student 3''': [[User:wonderland|Ya He]]<br />
*'''Student 4''': [[User:viva8565 | Sheng Feng]]<br />
*'''Student 5''': [[User:Forrest_Bao|Forrest Sheng Bao]]<br />
<br />
|<br />
<gallery><br />
Image:Forrest.JPG| [[User:Forrest_Bao|Forrest Sheng Bao]]<br />
Image:Sally.jpg|[[User:Sally730|Jingyi Wang]]<br />
Image:Jenny.jpg|[[User:wonderland|Ya He]]<br />
Image:Team_member_4.png|[[User:proline|Gang Wu]]<br />
Image:jason.jpg|[[User:viva8565 | Sheng Feng]]<br />
Image:Lavi.jpg|Shi Chen<br />
</gallery><br />
|}<br />
<br />
== Where we are from ==<br />
[[User:Forrest_Bao|Forrest Sheng Bao]] is from Dept. of Computer Science and Dept. of Electrical Engineering, Texas Tech University, Texas. He is a master student in Electrical Engineering and PhD candidate in Computer Science. He obtained a bachelor degree in electrical engineering in 2006.<br />
<br />
[[User:Sally730|Jingyi Wang]] is a senior of environmental science and technology, Beijing Normal University.<br />
<br />
[[User:wonderland|Ya He]] is a fourth year student studying Environmental engineering at Beijing Normal University. She had previously spent most of her childhood in Oxford, UK.<br />
<br />
[[User:proline | Gang Wu]] graduated from Department of biochemsitry, Nanjing University in 2006.<br />
<br />
[[User:viva8565 | Sheng Feng]] is a master student from College of Life science, Beijing Normal University.<br />
<br />
Shi Chen is Ph.D candidate of Entomology at Penn. State Univ. Currently he is also a Ph.D dual-degree student majoring Operations Research. His research interests are broad and include animal predation behavior, insect life history analysis, integrated pest management, insect wing venation analysis and water pollution prediction, etc. He likes to use mathematical, statistical and computational techniques to solve real world problems. His extracurricular interests include classical literature, music and photography. He really cherishes this chance of iGEM that I could work with many friends and have great communication with them.<br />
<br />
== What we have done ==<br />
===Forrest===<br />
[[User:Forrest_Bao|Forrest Sheng Bao]]'s research interests are in artificial intelligence, biomedical engineering and bioinformatics. He is very interested on algorithms in biology and medicine fields, as well as the interdisciplinary fields of computing and biology.<br />
<br />
This is his<br />
<html><br />
publications:</span><br />
<div style="margin-left: 10px;"><br />
<ul><br />
<li><u>Forrest Sheng Bao</u>, <i>et al.</i>, Automated Recognition and Diagnosis of Epilepsy Using EEG and a Probabilistic Neural Network, <i>20th IEEE Int'l Conference on Tools with Artificial Intelligence (ICTAI 2008)</i>, Nov. 2008, To appear</li><br />
<li>Lei Yuan and <u>Forrest Sheng Bao</u>, Automatic Analysis of Facial Expressions of Acute Pain in Neonates Using Boosted Gabor Features, <i>20th IEEE Int'l Conference on Tools with Artificial Intelligence (ICTAI 2008)</i>, Nov. 2008, To appear<br />
<li>Yijin Zhang, <u>Forrest Sheng Bao</u>, <i>et al</i>., Analysis of Energy Efficiency and Power Saving in IEEE 802.15.4, <i>IEEE WCNC 2007</i>, 2007</li><br />
<li>Yu-Xuan Wang and <u>Forrest Sheng Bao</u>, An Entropy-based Weighted Clustering Algorithm and Its Optimization for Ad hoc Networks, <i>IEEE WiMob 2007</i>, 2007</li><br />
<li>Stephen Gang Wu, <u>Forrest Sheng Bao</u>, Eric You Xu, Yu-Xuan Wang, Yi-Fan Chang and Chiao-Liang Shiang, A Leaf Recognition Algorithm for Plant classification Using Probabilistic Neural Network, <i>IEEE ISSPIT 2007</i>, 2007</li><br />
<li>Zhendong Zhao, Lei Yuan, Yuxuan Wang, <u>Forrest Sheng Bao</u>,A Novel Model of Working Set Selection for SMO Decomposition Methods, <i>IEEE ICTAI 2007</i>, 2007</li><br />
<br />
</ul><br />
</div><br />
</html><br />
<br />
===Jingyi===<br />
<br />
As a new graduate, [[User:Sally730 | Jingyi Wang]] does not have much experience in research and the program. From Nov. 2007 till now, she is working on the project "Detection of chlorophenols in aqueous phase using an Enzyme Thermistor", which is a China national 863 program. She does research on a laccase enzyme column based biosensor using thermometric measurement to detect chlorophenals pollutants. From May 2007 to Dec. 2007, she was involved in the project "Analysis of the distribution of PAHs in the conservation zone of Huang He estuary", which is a China national 973 project. Her job in the project is sampling, extracting, detecting and analysis PAHs in aqueous phase and solid phase, as well as assessing the ecological risk of the region.<br />
<br />
===Gang===<br />
<br />
[[User:proline|Gang Wu]] holds a China software patent "An Automatic System of Leaf Recognition" (NO.2006118213) and has one publication "A Leaf Recognition Algorithm for Plant Classification Using Probabilistic Neural Network", IEEE International Symposium on Signal Processing and Information Technology 2007, p12 - 17, 2007.<br />
<br />
From Mar. 2007 till now, he is working on the project "Self-regulatory gene network and new components for gene regulation", His job is to implement the classical Lac regulatory system to design a self-repression genetic switch. Learn the properties of sRNA in regulatory circuit. During Aug.2005 to Sept.2005 and Mar.2007, he utilized an SIRS model to analyze the pollution and decontamination information of past ten years and predict the tendency and quality of water in next twenty years in the project "A water pollution and decontamination model for Yangtze River". From Mar. 2005 to Jun. 2005, he was involved in the project "Microbial degradation of PU (polyurethane)". He collected and selected PU degradation microbes from environment using classical method of microbiology, participator.<br />
<br />
===Ya===<br />
Ya's previous research experience includes chemical engineering and supply chain optimization. She participated in the Hi-tech Research and Development Program of China (No. 2006AA06Z323): Investigating New Methods Using Laccase as Photocatalyst to Degrade Chlorophenols. She was mainly investigating photocatalysis and the synergistic effect of different catalysis reacting in one system. She has also been engaged in research adopting the system approach to achieve the most optimum and sustainable utility of supply chain. <br />
<br />
===Sheng===<br />
From Sept. 2007 till now, [[User:viva8565| Sheng]] is working on the project "Proteomic Study on Progression and Metastasis of Cancer", directed by Prof. Xueyuan Xiao (Universitis’ Conferderated Institute of Proteomics, Beijing Normal University) to study the tumor bio-markers and their function in progression and metastasis of cancer through proteomics method.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/TeamTeam:Beijing Normal/Team2008-10-30T00:24:55Z<p>Sally730: /* Who we are */</p>
<hr />
<div>__NOTOC__<br />
{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== This is Team Beijing Normal ==<br />
<br />
[[Image:Example_bnu logo.png]] <br />
<br />
The logo above is ours. The idea behind the design is that you can see us from a microscope, and it has been magnified 400 times. The person in the middle of the cell is <b>i</b>, one of us. The bulb is our idea, inside a cell.<br />
<br />
[[Image:team bnu.jpg|thumb|720px|center|Team members in China. From left: Ya, Jingyi, Sheng, Gang]]<br />
<br />
==Our lab==<br />
<gallery><br />
Image: BNUlab001.jpg<br />
Image: BNUlab002.jpg<br />
Image: BNUlab003.jpg<br />
Image: BNUlab004.jpg<br />
Image: BNUlab005.jpg<br />
Image: BNUlab006.jpg<br />
Image: BNUlab007.jpg<br />
Image: BNUlab008.jpg<br />
</gallery><br />
<br />
== Who we are ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
<br />
'''Advisors:'''<br />
<br />
*''' Advisor 1''': Junfeng Niu<br />
*'''Advisor 2''': Shi Chen<br />
<br />
'''Students:'''<br />
<br />
*'''Student 1''': [[User:proline|Gang Wu]]<br />
*'''Student 2''': [[User:Sally730|Jingyi Wang]]<br />
*'''Student 3''': [[User:wonderland|Ya He]]<br />
*'''Student 4''': [[User:viva8565 | Sheng Feng]]<br />
*'''Student 5''': [[User:Forrest_Bao|Forrest Sheng Bao]]<br />
<br />
|<br />
<gallery><br />
Image:Forrest.JPG| [[User:Forrest_Bao|Forrest Sheng Bao]]<br />
Image:Sally.jpg|[[User:Sally730|Jingyi Wang]]<br />
Image:Jenny.jpg|[[User:wonderland|Ya He]]<br />
Image:Team_member_4.png|[[User:proline|Gang Wu]]<br />
Image:jason.png|[[User:viva8565 | Sheng Feng]]<br />
Image:Lavi.jpg|Shi Chen<br />
</gallery><br />
|}<br />
<br />
== Where we are from ==<br />
[[User:Forrest_Bao|Forrest Sheng Bao]] is from Dept. of Computer Science and Dept. of Electrical Engineering, Texas Tech University, Texas. He is a master student in Electrical Engineering and PhD candidate in Computer Science. He obtained a bachelor degree in electrical engineering in 2006.<br />
<br />
[[User:Sally730|Jingyi Wang]] is a senior of environmental science and technology, Beijing Normal University.<br />
<br />
[[User:wonderland|Ya He]] is a fourth year student studying Environmental engineering at Beijing Normal University. She had previously spent most of her childhood in Oxford, UK.<br />
<br />
[[User:proline | Gang Wu]] graduated from Department of biochemsitry, Nanjing University in 2006.<br />
<br />
[[User:viva8565 | Sheng Feng]] is a master student from College of Life science, Beijing Normal University.<br />
<br />
Shi Chen is Ph.D candidate of Entomology at Penn. State Univ. Currently he is also a Ph.D dual-degree student majoring Operations Research. His research interests are broad and include animal predation behavior, insect life history analysis, integrated pest management, insect wing venation analysis and water pollution prediction, etc. He likes to use mathematical, statistical and computational techniques to solve real world problems. His extracurricular interests include classical literature, music and photography. He really cherishes this chance of iGEM that I could work with many friends and have great communication with them.<br />
<br />
== What we have done ==<br />
===Forrest===<br />
[[User:Forrest_Bao|Forrest Sheng Bao]]'s research interests are in artificial intelligence, biomedical engineering and bioinformatics. He is very interested on algorithms in biology and medicine fields, as well as the interdisciplinary fields of computing and biology.<br />
<br />
This is his<br />
<html><br />
publications:</span><br />
<div style="margin-left: 10px;"><br />
<ul><br />
<li><u>Forrest Sheng Bao</u>, <i>et al.</i>, Automated Recognition and Diagnosis of Epilepsy Using EEG and a Probabilistic Neural Network, <i>20th IEEE Int'l Conference on Tools with Artificial Intelligence (ICTAI 2008)</i>, Nov. 2008, To appear</li><br />
<li>Lei Yuan and <u>Forrest Sheng Bao</u>, Automatic Analysis of Facial Expressions of Acute Pain in Neonates Using Boosted Gabor Features, <i>20th IEEE Int'l Conference on Tools with Artificial Intelligence (ICTAI 2008)</i>, Nov. 2008, To appear<br />
<li>Yijin Zhang, <u>Forrest Sheng Bao</u>, <i>et al</i>., Analysis of Energy Efficiency and Power Saving in IEEE 802.15.4, <i>IEEE WCNC 2007</i>, 2007</li><br />
<li>Yu-Xuan Wang and <u>Forrest Sheng Bao</u>, An Entropy-based Weighted Clustering Algorithm and Its Optimization for Ad hoc Networks, <i>IEEE WiMob 2007</i>, 2007</li><br />
<li>Stephen Gang Wu, <u>Forrest Sheng Bao</u>, Eric You Xu, Yu-Xuan Wang, Yi-Fan Chang and Chiao-Liang Shiang, A Leaf Recognition Algorithm for Plant classification Using Probabilistic Neural Network, <i>IEEE ISSPIT 2007</i>, 2007</li><br />
<li>Zhendong Zhao, Lei Yuan, Yuxuan Wang, <u>Forrest Sheng Bao</u>,A Novel Model of Working Set Selection for SMO Decomposition Methods, <i>IEEE ICTAI 2007</i>, 2007</li><br />
<br />
</ul><br />
</div><br />
</html><br />
<br />
===Jingyi===<br />
<br />
As a new graduate, [[User:Sally730 | Jingyi Wang]] does not have much experience in research and the program. From Nov. 2007 till now, she is working on the project "Detection of chlorophenols in aqueous phase using an Enzyme Thermistor", which is a China national 863 program. She does research on a laccase enzyme column based biosensor using thermometric measurement to detect chlorophenals pollutants. From May 2007 to Dec. 2007, she was involved in the project "Analysis of the distribution of PAHs in the conservation zone of Huang He estuary", which is a China national 973 project. Her job in the project is sampling, extracting, detecting and analysis PAHs in aqueous phase and solid phase, as well as assessing the ecological risk of the region.<br />
<br />
===Gang===<br />
<br />
[[User:proline|Gang Wu]] holds a China software patent "An Automatic System of Leaf Recognition" (NO.2006118213) and has one publication "A Leaf Recognition Algorithm for Plant Classification Using Probabilistic Neural Network", IEEE International Symposium on Signal Processing and Information Technology 2007, p12 - 17, 2007.<br />
<br />
From Mar. 2007 till now, he is working on the project "Self-regulatory gene network and new components for gene regulation", His job is to implement the classical Lac regulatory system to design a self-repression genetic switch. Learn the properties of sRNA in regulatory circuit. During Aug.2005 to Sept.2005 and Mar.2007, he utilized an SIRS model to analyze the pollution and decontamination information of past ten years and predict the tendency and quality of water in next twenty years in the project "A water pollution and decontamination model for Yangtze River". From Mar. 2005 to Jun. 2005, he was involved in the project "Microbial degradation of PU (polyurethane)". He collected and selected PU degradation microbes from environment using classical method of microbiology, participator.<br />
<br />
===Ya===<br />
Ya's previous research experience includes chemical engineering and supply chain optimization. She participated in the Hi-tech Research and Development Program of China (No. 2006AA06Z323): Investigating New Methods Using Laccase as Photocatalyst to Degrade Chlorophenols. She was mainly investigating photocatalysis and the synergistic effect of different catalysis reacting in one system. She has also been engaged in research adopting the system approach to achieve the most optimum and sustainable utility of supply chain. <br />
<br />
===Sheng===<br />
From Sept. 2007 till now, [[User:viva8565| Sheng]] is working on the project "Proteomic Study on Progression and Metastasis of Cancer", directed by Prof. Xueyuan Xiao (Universitis’ Conferderated Institute of Proteomics, Beijing Normal University) to study the tumor bio-markers and their function in progression and metastasis of cancer through proteomics method.</div>Sally730http://2008.igem.org/File:Team_member_5.pngFile:Team member 5.png2008-10-30T00:22:47Z<p>Sally730: uploaded a new version of "Image:Team member 5.png": Reverted to version as of 02:04, 17 October 2008</p>
<hr />
<div></div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T00:15:58Z<p>Sally730: /* design abstract */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', Vol. 105, No. 5, 433–449. 2008, permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== Design Abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal.<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
<br />
<br />
== References for special protocol: ==<br />
1.Simplified hot start PCR, David E. Birch et al. Nature 381:445-446 (1996)<br />
<br />
2.An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose, S.Kaijalainen et al. Nucleic Acids Research 21:2959-2960 (1993)<br />
<br />
3.Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta)gene, Yukihiro Kitade et al. Journal of Applied Phycology 15:555-557 (2003)<br />
<br />
4.An efficient and economic enhancer mix for PCR, Markus Ralser et al. Biochemical and Biophysical Research Communications 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
Design details<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
1. Switch on the degradation pathway<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
2. Solve the bottleneck in PCBs degradation pathway<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
3. Control on the amount of PCBs molecular that enter the cell.<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
4. System guarantee<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
Reference: <br />
<br />
[1] Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features. Kensuke Furukawa and Hidehiko Fujihara, Journal of bioscience and bioengineering, 2008, Vol. 105, No. 5:433–449,<br />
<br />
[2] Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls. Seah S. Y., Labbe, G., Kaschabek, S. R., Reifenrath, F.,<br />
Reineke, F., and Eltis, L. D., J. Bacteriol., 2001, 183:1511–1516.<br />
<br />
[3] Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants. Sang-ho Park,Young Lee, Jong-Chan Chae and Chi-Kyung Kim. Environmental Monitoring and Assessment, 2004, 92:241–251.<br />
<br />
[4] Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery. Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard, Yongchun Tang. Biotechnology and Bioengineering, 2007, Vol. 98, No. 4.<br />
<br />
[5] ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls. Jessika Feliciano, Shifen Xu, Xiyuan Guan, Hans-Joachim Lehmle , Leonidas G. Bachas, Sylvia Daunert. Anal Bioanal Chem, 2006, 385: 807–813<br />
<br />
== Results ==<br />
1. Protocols<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
2. Plasmids constructed<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
3. A functional part <br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector. <br />
<br />
<br />
== Acknowledgement ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* David N. Dowling<br />
<br />
For the bacteria E. coil SMl0.<br />
<br />
* Professor Niu Junfeng<br />
<br />
For his instructions and advices<br />
<br />
* Tsinghua Team <br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* Chiba Team<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* Tokyo Tech Team<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* USTC Team<br />
<br />
For suggestions on our experiments and delicious food in Hefei.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T00:12:24Z<p>Sally730: /* acknowledgement */</p>
<hr />
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!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', Vol. 105, No. 5, 433–449. 2008, permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== design abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal. <br />
<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
<br />
<br />
== References for special protocol: ==<br />
1.Simplified hot start PCR, David E. Birch et al. Nature 381:445-446 (1996)<br />
<br />
2.An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose, S.Kaijalainen et al. Nucleic Acids Research 21:2959-2960 (1993)<br />
<br />
3.Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta)gene, Yukihiro Kitade et al. Journal of Applied Phycology 15:555-557 (2003)<br />
<br />
4.An efficient and economic enhancer mix for PCR, Markus Ralser et al. Biochemical and Biophysical Research Communications 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
Design details<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
1. Switch on the degradation pathway<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
2. Solve the bottleneck in PCBs degradation pathway<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
3. Control on the amount of PCBs molecular that enter the cell.<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
4. System guarantee<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
Reference: <br />
<br />
[1] Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features. Kensuke Furukawa and Hidehiko Fujihara, Journal of bioscience and bioengineering, 2008, Vol. 105, No. 5:433–449,<br />
<br />
[2] Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls. Seah S. Y., Labbe, G., Kaschabek, S. R., Reifenrath, F.,<br />
Reineke, F., and Eltis, L. D., J. Bacteriol., 2001, 183:1511–1516.<br />
<br />
[3] Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants. Sang-ho Park,Young Lee, Jong-Chan Chae and Chi-Kyung Kim. Environmental Monitoring and Assessment, 2004, 92:241–251.<br />
<br />
[4] Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery. Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard, Yongchun Tang. Biotechnology and Bioengineering, 2007, Vol. 98, No. 4.<br />
<br />
[5] ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls. Jessika Feliciano, Shifen Xu, Xiyuan Guan, Hans-Joachim Lehmle , Leonidas G. Bachas, Sylvia Daunert. Anal Bioanal Chem, 2006, 385: 807–813<br />
<br />
== Results ==<br />
1. Protocols<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
2. Plasmids constructed<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
3. A functional part <br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector. <br />
<br />
<br />
== Acknowledgement ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* David N. Dowling<br />
<br />
For the bacteria E. coil SMl0.<br />
<br />
* Professor Niu Junfeng<br />
<br />
For his instructions and advices<br />
<br />
* Tsinghua Team <br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* Chiba Team<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* Tokyo Tech Team<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* USTC Team<br />
<br />
For suggestions on our experiments and delicious food in Hefei.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T00:10:09Z<p>Sally730: /* Design Details */</p>
<hr />
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!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', Vol. 105, No. 5, 433–449. 2008, permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== design abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal. <br />
<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
<br />
<br />
== References for special protocol: ==<br />
1.Simplified hot start PCR, David E. Birch et al. Nature 381:445-446 (1996)<br />
<br />
2.An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose, S.Kaijalainen et al. Nucleic Acids Research 21:2959-2960 (1993)<br />
<br />
3.Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta)gene, Yukihiro Kitade et al. Journal of Applied Phycology 15:555-557 (2003)<br />
<br />
4.An efficient and economic enhancer mix for PCR, Markus Ralser et al. Biochemical and Biophysical Research Communications 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
Design details<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
1. Switch on the degradation pathway<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system is awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
2. Solve the bottleneck in PCBs degradation pathway<br />
<br />
Our idea: Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
3. Control on the amount of PCBs molecular that enter the cell.<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
4. System guarantee<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
Reference: <br />
<br />
[1] Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features. Kensuke Furukawa and Hidehiko Fujihara, Journal of bioscience and bioengineering, 2008, Vol. 105, No. 5:433–449,<br />
<br />
[2] Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls. Seah S. Y., Labbe, G., Kaschabek, S. R., Reifenrath, F.,<br />
Reineke, F., and Eltis, L. D., J. Bacteriol., 2001, 183:1511–1516.<br />
<br />
[3] Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants. Sang-ho Park,Young Lee, Jong-Chan Chae and Chi-Kyung Kim. Environmental Monitoring and Assessment, 2004, 92:241–251.<br />
<br />
[4] Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery. Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard, Yongchun Tang. Biotechnology and Bioengineering, 2007, Vol. 98, No. 4.<br />
<br />
[5] ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls. Jessika Feliciano, Shifen Xu, Xiyuan Guan, Hans-Joachim Lehmle , Leonidas G. Bachas, Sylvia Daunert. Anal Bioanal Chem, 2006, 385: 807–813<br />
<br />
== Results ==<br />
1. Protocols<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
2. Plasmids constructed<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
3. A functional part <br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector. <br />
<br />
<br />
== acknowledgement ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* David N. Dowling<br />
<br />
For the bacteria E. coil SMl0.<br />
<br />
* Professor Niu Junfeng<br />
<br />
For his instructions and advices<br />
<br />
* Tsinghua Team <br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* Chiba Team<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* Tokyo Tech Team<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* USTC Team<br />
<br />
For suggestions on our experiments and delicious food in Hefei.</div>Sally730http://2008.igem.org/File:T4.jpgFile:T4.jpg2008-10-30T00:07:47Z<p>Sally730: </p>
<hr />
<div></div>Sally730http://2008.igem.org/File:T3.jpgFile:T3.jpg2008-10-30T00:06:54Z<p>Sally730: </p>
<hr />
<div></div>Sally730http://2008.igem.org/File:T2.jpgFile:T2.jpg2008-10-30T00:06:06Z<p>Sally730: </p>
<hr />
<div></div>Sally730http://2008.igem.org/File:Pump.gifFile:Pump.gif2008-10-30T00:04:29Z<p>Sally730: </p>
<hr />
<div></div>Sally730http://2008.igem.org/File:T1.jpgFile:T1.jpg2008-10-30T00:03:21Z<p>Sally730: </p>
<hr />
<div></div>Sally730http://2008.igem.org/File:Awake.gifFile:Awake.gif2008-10-30T00:01:47Z<p>Sally730: </p>
<hr />
<div></div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-30T00:00:58Z<p>Sally730: /* Design Details */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', Vol. 105, No. 5, 433–449. 2008, permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== design abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal. <br />
<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
<br />
<br />
== References for special protocol: ==<br />
1.Simplified hot start PCR, David E. Birch et al. Nature 381:445-446 (1996)<br />
<br />
2.An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose, S.Kaijalainen et al. Nucleic Acids Research 21:2959-2960 (1993)<br />
<br />
3.Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta)gene, Yukihiro Kitade et al. Journal of Applied Phycology 15:555-557 (2003)<br />
<br />
4.An efficient and economic enhancer mix for PCR, Markus Ralser et al. Biochemical and Biophysical Research Communications 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
Design details<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
1. Switch on the degradation pathway<br />
<br />
[[Image:awake.gif]]<br />
<br />
The system was awakened by PCBs.<br />
<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:t1.jpg]] <br />
<br />
<br />
2. Solve the bottleneck in PCBs degradation pathway<br />
<br />
Add a spur track to solve the bottleneck of the pump.<br />
<br />
[[Image:pump.gif]]<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:t2.jpg]]<br />
<br />
3. Control on the amount of PCBs molecular that enter the cell.<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:t3.jpg]]<br />
<br />
4. System guarantee<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:t4.jpg]]<br />
<br />
<br />
Reference: <br />
<br />
[1] Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features. Kensuke Furukawa and Hidehiko Fujihara, Journal of bioscience and bioengineering, 2008, Vol. 105, No. 5:433–449,<br />
<br />
[2] Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls. Seah S. Y., Labbe, G., Kaschabek, S. R., Reifenrath, F.,<br />
Reineke, F., and Eltis, L. D., J. Bacteriol., 2001, 183:1511–1516.<br />
<br />
[3] Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants. Sang-ho Park,Young Lee, Jong-Chan Chae and Chi-Kyung Kim. Environmental Monitoring and Assessment, 2004, 92:241–251.<br />
<br />
[4] Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery. Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard, Yongchun Tang. Biotechnology and Bioengineering, 2007, Vol. 98, No. 4.<br />
<br />
[5] ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls. Jessika Feliciano, Shifen Xu, Xiyuan Guan, Hans-Joachim Lehmle , Leonidas G. Bachas, Sylvia Daunert. Anal Bioanal Chem, 2006, 385: 807–813<br />
<br />
== Results ==<br />
1. Protocols<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
2. Plasmids constructed<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
3. A functional part <br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector. <br />
<br />
<br />
== acknowledgement ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* David N. Dowling<br />
<br />
For the bacteria E. coil SMl0.<br />
<br />
* Professor Niu Junfeng<br />
<br />
For his instructions and advices<br />
<br />
* Tsinghua Team <br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* Chiba Team<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* Tokyo Tech Team<br />
<br />
For all the information and advice and encouragement.<br />
<br />
* USTC Team<br />
<br />
For suggestions on our experiments and delicious food in Hefei.</div>Sally730http://2008.igem.org/File:S4.jpgFile:S4.jpg2008-10-29T11:56:07Z<p>Sally730: </p>
<hr />
<div></div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-29T11:55:37Z<p>Sally730: /* Design Details */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', Vol. 105, No. 5, 433–449. 2008, permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== design abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal. <br />
<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
<br />
<br />
== References for special protocol: ==<br />
1.Simplified hot start PCR, David E. Birch et al. Nature 381:445-446 (1996)<br />
<br />
2.An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose, S.Kaijalainen et al. Nucleic Acids Research 21:2959-2960 (1993)<br />
<br />
3.Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta)gene, Yukihiro Kitade et al. Journal of Applied Phycology 15:555-557 (2003)<br />
<br />
4.An efficient and economic enhancer mix for PCR, Markus Ralser et al. Biochemical and Biophysical Research Communications 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
Design details<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
1. Switch on the degradation pathway<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:scheme1.jpg]] <br />
<br />
<br />
2. Solve the bottleneck in PCBs degradation pathway<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:s2.jpg]]<br />
<br />
3. Control on the amount of PCBs molecular that enter the cell.<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:pp3.jpg]]<br />
<br />
4. System guarantee<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:s4.jpg]]<br />
<br />
<br />
Reference: <br />
<br />
[1] Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features. Kensuke Furukawa and Hidehiko Fujihara, Journal of bioscience and bioengineering, 2008, Vol. 105, No. 5:433–449,<br />
<br />
[2] Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls. Seah S. Y., Labbe, G., Kaschabek, S. R., Reifenrath, F.,<br />
Reineke, F., and Eltis, L. D., J. Bacteriol., 2001, 183:1511–1516.<br />
<br />
[3] Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants. Sang-ho Park,Young Lee, Jong-Chan Chae and Chi-Kyung Kim. Environmental Monitoring and Assessment, 2004, 92:241–251.<br />
<br />
[4] Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery. Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard, Yongchun Tang. Biotechnology and Bioengineering, 2007, Vol. 98, No. 4.<br />
<br />
[5] ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls. Jessika Feliciano, Shifen Xu, Xiyuan Guan, Hans-Joachim Lehmle , Leonidas G. Bachas, Sylvia Daunert. Anal Bioanal Chem, 2006, 385: 807–813<br />
<br />
== Results ==<br />
1. Protocols<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
2. Plasmids constructed<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
3. A functional part <br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector. <br />
<br />
<br />
== acknowledgement ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* David N. Dowling<br />
<br />
For the bacteria E. coil Sml0.<br />
<br />
* Professor Niu Junfeng<br />
<br />
For his instructions and advices<br />
<br />
* Tsinghua Team <br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* Chiba Team<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* Tokyo Tech Team<br />
<br />
For all the information and advice and encouragement.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-29T11:52:11Z<p>Sally730: /* Design Details */</p>
<hr />
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!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', Vol. 105, No. 5, 433–449. 2008, permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== design abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal. <br />
<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
<br />
<br />
== References for special protocol: ==<br />
1.Simplified hot start PCR, David E. Birch et al. Nature 381:445-446 (1996)<br />
<br />
2.An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose, S.Kaijalainen et al. Nucleic Acids Research 21:2959-2960 (1993)<br />
<br />
3.Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta)gene, Yukihiro Kitade et al. Journal of Applied Phycology 15:555-557 (2003)<br />
<br />
4.An efficient and economic enhancer mix for PCR, Markus Ralser et al. Biochemical and Biophysical Research Communications 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
Design details<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
1. Switch on the degradation pathway<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:scheme1.jpg]] <br />
<br />
<br />
2. Solve the bottleneck in PCBs degradation pathway<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:s2.jpg]]<br />
<br />
3. Control on the amount of PCBs molecular that enter the cell.<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:pp3.jpg]]<br />
<br />
4. System guarantee<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:p4.jpg]]<br />
<br />
<br />
Reference: <br />
<br />
[1] Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features. Kensuke Furukawa and Hidehiko Fujihara, Journal of bioscience and bioengineering, 2008, Vol. 105, No. 5:433–449,<br />
<br />
[2] Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls. Seah S. Y., Labbe, G., Kaschabek, S. R., Reifenrath, F.,<br />
Reineke, F., and Eltis, L. D., J. Bacteriol., 2001, 183:1511–1516.<br />
<br />
[3] Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants. Sang-ho Park,Young Lee, Jong-Chan Chae and Chi-Kyung Kim. Environmental Monitoring and Assessment, 2004, 92:241–251.<br />
<br />
[4] Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery. Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard, Yongchun Tang. Biotechnology and Bioengineering, 2007, Vol. 98, No. 4.<br />
<br />
[5] ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls. Jessika Feliciano, Shifen Xu, Xiyuan Guan, Hans-Joachim Lehmle , Leonidas G. Bachas, Sylvia Daunert. Anal Bioanal Chem, 2006, 385: 807–813<br />
<br />
== Results ==<br />
1. Protocols<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
2. Plasmids constructed<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
3. A functional part <br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector. <br />
<br />
<br />
== acknowledgement ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* David N. Dowling<br />
<br />
For the bacteria E. coil Sml0.<br />
<br />
* Professor Niu Junfeng<br />
<br />
For his instructions and advices<br />
<br />
* Tsinghua Team <br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* Chiba Team<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* Tokyo Tech Team<br />
<br />
For all the information and advice and encouragement.</div>Sally730http://2008.igem.org/File:Pp3.jpgFile:Pp3.jpg2008-10-29T11:49:48Z<p>Sally730: </p>
<hr />
<div></div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-29T11:49:28Z<p>Sally730: /* Design Details */</p>
<hr />
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!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', Vol. 105, No. 5, 433–449. 2008, permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== design abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal. <br />
<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
<br />
<br />
== References for special protocol: ==<br />
1.Simplified hot start PCR, David E. Birch et al. Nature 381:445-446 (1996)<br />
<br />
2.An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose, S.Kaijalainen et al. Nucleic Acids Research 21:2959-2960 (1993)<br />
<br />
3.Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta)gene, Yukihiro Kitade et al. Journal of Applied Phycology 15:555-557 (2003)<br />
<br />
4.An efficient and economic enhancer mix for PCR, Markus Ralser et al. Biochemical and Biophysical Research Communications 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
Design details<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
1. Switch on the degradation pathway<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:scheme1.jpg]] <br />
<br />
<br />
2. Solve the bottleneck in PCBs degradation pathway<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:s2.jpg]]<br />
<br />
3. Control on the amount of PCBs molecular that enter the cell.<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:pp3.jpg]]<br />
<br />
4. System guarantee<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:s4.jpg]]<br />
<br />
<br />
Reference: <br />
<br />
[1] Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features. Kensuke Furukawa and Hidehiko Fujihara, Journal of bioscience and bioengineering, 2008, Vol. 105, No. 5:433–449,<br />
<br />
[2] Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls. Seah S. Y., Labbe, G., Kaschabek, S. R., Reifenrath, F.,<br />
Reineke, F., and Eltis, L. D., J. Bacteriol., 2001, 183:1511–1516.<br />
<br />
[3] Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants. Sang-ho Park,Young Lee, Jong-Chan Chae and Chi-Kyung Kim. Environmental Monitoring and Assessment, 2004, 92:241–251.<br />
<br />
[4] Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery. Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard, Yongchun Tang. Biotechnology and Bioengineering, 2007, Vol. 98, No. 4.<br />
<br />
[5] ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls. Jessika Feliciano, Shifen Xu, Xiyuan Guan, Hans-Joachim Lehmle , Leonidas G. Bachas, Sylvia Daunert. Anal Bioanal Chem, 2006, 385: 807–813<br />
<br />
== Results ==<br />
1. Protocols<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
2. Plasmids constructed<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
3. A functional part <br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector. <br />
<br />
<br />
== acknowledgement ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* David N. Dowling<br />
<br />
For the bacteria E. coil Sml0.<br />
<br />
* Professor Niu Junfeng<br />
<br />
For his instructions and advices<br />
<br />
* Tsinghua Team <br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* Chiba Team<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* Tokyo Tech Team<br />
<br />
For all the information and advice and encouragement.</div>Sally730http://2008.igem.org/File:S3.jpgFile:S3.jpg2008-10-29T11:48:44Z<p>Sally730: uploaded a new version of "Image:S3.jpg"</p>
<hr />
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<hr />
<div></div>Sally730http://2008.igem.org/File:S3.jpgFile:S3.jpg2008-10-29T11:47:03Z<p>Sally730: uploaded a new version of "Image:S3.jpg"</p>
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<div></div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-29T11:16:50Z<p>Sally730: /* Results */</p>
<hr />
<div>{| style="color:#ffffff;background-color:#6699cc;" cellpadding="3" cellspacing="1" border="0" bordercolor="#fff" align="center" width="80%"<br />
!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', Vol. 105, No. 5, 433–449. 2008, permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== design abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal. <br />
<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
<br />
<br />
== References for special protocol: ==<br />
1.Simplified hot start PCR, David E. Birch et al. Nature 381:445-446 (1996)<br />
<br />
2.An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose, S.Kaijalainen et al. Nucleic Acids Research 21:2959-2960 (1993)<br />
<br />
3.Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta)gene, Yukihiro Kitade et al. Journal of Applied Phycology 15:555-557 (2003)<br />
<br />
4.An efficient and economic enhancer mix for PCR, Markus Ralser et al. Biochemical and Biophysical Research Communications 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
Design details<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
1. Switch on the degradation pathway<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:scheme1.jpg]] <br />
<br />
<br />
2. Solve the bottleneck in PCBs degradation pathway<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:s2.jpg]]<br />
<br />
3. Control on the amount of PCBs molecular that enter the cell.<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:s3.jpg]]<br />
<br />
4. System guarantee<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:p4.jpg]]<br />
<br />
<br />
Reference: <br />
<br />
[1] Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features. Kensuke Furukawa and Hidehiko Fujihara, Journal of bioscience and bioengineering, 2008, Vol. 105, No. 5:433–449,<br />
<br />
[2] Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls. Seah S. Y., Labbe, G., Kaschabek, S. R., Reifenrath, F.,<br />
Reineke, F., and Eltis, L. D., J. Bacteriol., 2001, 183:1511–1516.<br />
<br />
[3] Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants. Sang-ho Park,Young Lee, Jong-Chan Chae and Chi-Kyung Kim. Environmental Monitoring and Assessment, 2004, 92:241–251.<br />
<br />
[4] Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery. Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard, Yongchun Tang. Biotechnology and Bioengineering, 2007, Vol. 98, No. 4.<br />
<br />
[5] ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls. Jessika Feliciano, Shifen Xu, Xiyuan Guan, Hans-Joachim Lehmle , Leonidas G. Bachas, Sylvia Daunert. Anal Bioanal Chem, 2006, 385: 807–813<br />
<br />
== Results ==<br />
1. Protocols<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
2. Plasmids constructed<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
3. A functional part <br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector. <br />
<br />
<br />
== acknowledgement ==<br />
<br />
*Professor Rolf-Michael wittich and Professor Kenneth N. Timmis<br />
<br />
For the bacteria Sphingomonas sp. Strain RW1 they provided.<br />
<br />
* David N. Dowling<br />
<br />
For the bacteria E. coil Sml0.<br />
<br />
* Professor Niu Junfeng<br />
<br />
For his instructions and advices<br />
<br />
* Tsinghua Team <br />
<br />
For the experience we shared and the indispensable help.<br />
<br />
* Chiba Team<br />
<br />
For all the information we shared and all the joy we had, especially Mai and Yoshimi.<br />
<br />
* Tokyo Tech Team<br />
<br />
For all the information and advice and encouragement.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-28T12:54:40Z<p>Sally730: /* Results */</p>
<hr />
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!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
!align="center"|[[Team:Beijing_Normal/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', Vol. 105, No. 5, 433–449. 2008, permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== design abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal. <br />
<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
<br />
*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
<br />
*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
<br />
*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
<br />
*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
<br />
== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
<br />
=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
<br />
After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
<br />
====Explanation====<br />
<br />
When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
<br />
===Effective Additives===<br />
<br />
The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
<br />
In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
<br />
==== Explanation ====<br />
<br />
One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
<br />
<br />
<br />
== References for special protocol: ==<br />
1.Simplified hot start PCR, David E. Birch et al. Nature 381:445-446 (1996)<br />
<br />
2.An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose, S.Kaijalainen et al. Nucleic Acids Research 21:2959-2960 (1993)<br />
<br />
3.Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta)gene, Yukihiro Kitade et al. Journal of Applied Phycology 15:555-557 (2003)<br />
<br />
4.An efficient and economic enhancer mix for PCR, Markus Ralser et al. Biochemical and Biophysical Research Communications 347:747–751 (2006)<br />
<br />
== Design Details ==<br />
Design details<br />
<br />
[[Image:p1.jpg]]<br />
<br />
Enhance the degradation efficiency & detect PCBs<br />
<br />
1. Switch on the degradation pathway<br />
<br />
We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
<br />
In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
<br />
[[Image:scheme1.jpg]] <br />
<br />
<br />
2. Solve the bottleneck in PCBs degradation pathway<br />
<br />
BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
<br />
[[Image:s2.jpg]]<br />
<br />
3. Control on the amount of PCBs molecular that enter the cell.<br />
<br />
As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
<br />
[[Image:s3.jpg]]<br />
<br />
4. System guarantee<br />
<br />
In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
<br />
[[Image:p4.jpg]]<br />
<br />
<br />
Reference: <br />
<br />
[1] Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features. Kensuke Furukawa and Hidehiko Fujihara, Journal of bioscience and bioengineering, 2008, Vol. 105, No. 5:433–449,<br />
<br />
[2] Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls. Seah S. Y., Labbe, G., Kaschabek, S. R., Reifenrath, F.,<br />
Reineke, F., and Eltis, L. D., J. Bacteriol., 2001, 183:1511–1516.<br />
<br />
[3] Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants. Sang-ho Park,Young Lee, Jong-Chan Chae and Chi-Kyung Kim. Environmental Monitoring and Assessment, 2004, 92:241–251.<br />
<br />
[4] Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery. Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard, Yongchun Tang. Biotechnology and Bioengineering, 2007, Vol. 98, No. 4.<br />
<br />
[5] ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls. Jessika Feliciano, Shifen Xu, Xiyuan Guan, Hans-Joachim Lehmle , Leonidas G. Bachas, Sylvia Daunert. Anal Bioanal Chem, 2006, 385: 807–813<br />
<br />
== Results ==<br />
1. Protocols<br />
<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
<br />
We have shared experience with several other teams, and comunicated a lot.<br />
<br />
2. Plasmids constructed<br />
<br />
We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
<br />
3. A functional part <br />
<br />
Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector.</div>Sally730http://2008.igem.org/Team:Beijing_Normal/ProjectTeam:Beijing Normal/Project2008-10-28T12:36:41Z<p>Sally730: /* Results */</p>
<hr />
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!align="center"|[[Team:Beijing_Normal|Home]]<br />
!align="center"|[[Team:Beijing_Normal/Team|The Team]]<br />
!align="center"|[[Team:Beijing_Normal/Project|The Project]]<br />
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!align="center"|[[Team:Beijing_Normal/Modeling|Modeling]]<br />
!align="center"|[[Team:Beijing_Normal/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Overall''' ==<br />
<br />
We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.<br />
<br />
Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.<br />
<br />
[[Image:pcbs.gif]]<br />
<br />
The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.<br />
<br />
Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.<br />
<br />
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have<br />
been the subject of great public and scientific scrutiny.<br />
<br />
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.<br />
<br />
Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.<br />
<br />
Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.<br />
<br />
Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way. And do some work on increasing the degradation effeciency.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== background information ===<br />
<br />
<br />
*Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.<br />
<br />
*Catabolic pathway for degradation of biphenyl and organization of bph gene cluster is as follows:<br />
[[Image:PCBs pathway.jpg|900px|center|thumb|Source: Kensuke Furukawa and Hidehiko Fujihara, '''Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features''', ''Journal of bioscience and bioengineering'', Vol. 105, No. 5, 433–449. 2008, permitted to use by Kensuke Furukawa]]<br />
<br />
'''Enzymes responsible for oxidative degradation of PCBs'''<br />
<br />
1 BphA: biphenyl dioxygenase<br />
<br />
Biphenyl dioxygenase is a Riesketype three-component enzyme, comprising a terminal dioxygenase that is composed of a large subunit (encoded by bphA1) and a small subunit (encoded by bphA2), a ferredoxin (encoded by bphA3) and a ferredoxin reductase (encodedby bphA4)<br />
<br />
It catalyzed the conversion of biphenyl to dihydrodiol compound in step 1.<br />
<br />
2 BphB: dihydriol dehydrogenase<br />
<br />
In the second step of the upper pathway BphB enzymes catalyzes the conversion of dihidrodiol to dihydroxy compound.<br />
<br />
3 BphC: 2,3-dihydroxybiphnyl dioxygenase<br />
<br />
The third enzyme in upper pathway is a 2,3-dihyroxybiphnyl dioxygenase involved in the ring meta-cleavage at the 1,2 position.<br />
<br />
4 BphD: hydrolase <br />
<br />
In the forth step BphD, a hydrolase, hydrolyzes the ring meta-cleavage yellow compound(HOPDA) to chlorobenzoic acid and 2-hydroxypenta-2,4-dienoate.<br />
<br />
=== design abstract ===<br />
<br />
Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Our task is to design an effective as well as intelligent PCBs degrader. Thus our work could be divided into two parts. One is to develop a sensing system for the detection of PCBs and activation of the downstream degradation pathway; the other is to enhance the degradation efficiency.<br />
<br />
DHBD(2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of DHBD, so we design a feedback activation pathway to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate Ppcb and thus increase gene expression downstream. <br />
<br />
As dihydrodiols and dihydroxybiphenyls are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. In this case, the amount of these two metabolites will be reduced in two ways.<br />
<br />
As to the sensor part, dihydroxylated PCBs are substrate of the clcA-encoded chlorocatechol dioxygenase and thus induce the clcR and related promoter, so we use this as the sensing system. And T7 amplification system is add downstream to amplify the signal. <br />
<br />
<br />
=== The Experiments ===<br />
*1.Get the parts from biobrick<br />
We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.<br />
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*2 PCR<br />
** 2.1 The pfu/Taq complex system<br />
Reagent Concentration/Activity 50ul in total <br />
taq buffer 10x 5 <br />
pfu/taq complex 0.8~1.0 <br />
dNTPmix 10mM each 4 <br />
Primer 1 10uM 1.5 <br />
Primer 2 10um 1.5 <br />
Template DNA changeable -- <br />
ddH2O --- add to 50ul <br />
** 2.2 The program under pfu/Tag complex system<br />
Progress Program I <br />
Predenaturing 95℃ 5 min <br />
Denaturing 95℃ 30sec <br />
Annealing (Tm-5) ℃ 30sec <br />
Extension 72℃ theoretically 1min/1kb<br />
Last extention 72℃ 5min<br />
Hold 4℃<br />
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*3 restriction enzyme digestion<br />
select suitable enzymes and buffer. <br />
Analyse the system using NEB cutter in case of double digestion. <br />
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp NEB cutter finder].<br />
37℃ water bath for 2~3h, 4h is preferable<br />
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*4 ligation<br />
The key to a successful ligation according to our experience is avoiding high temperature.<br />
After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more. <br />
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*5 transformation<br />
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.<br />
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== Special protocol ==<br />
To obtain some genes, we often have to use bacteria chromosome or large size plasmid as template in which high GC% content and complex secondary structures are seriously hampering PCR and thus leading to complete failure. To solve this problem, we have developed a special protocol-- hot start method combined with additive(sole or mixed)-- which is most helpful . <br />
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=== Simplified Hot Start PCR ===<br />
Hot start method is to prevent primer dimer and low specified product yield. In our experiment, We use common taq polymerase to replace commercial high-priced hot-start polymerase. <br />
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After a 5min 95℃ pre-heat step, DNA polymerase is added to each PCR tube before the PCR cycling begin. After that, the regular PCR cycling could begin.<br />
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====Explanation====<br />
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When reaction components are mixed at room temperature, reaction set up below the optimal primer annealing temperature, which permits nonspecific primer annealing and extention.Undesired, non-specific primer extetion products formed this way may be amplified in the PCR, resulting in misprimed products and primer ologomers.In hot start PCR, DNA polymerase is withheld from the mixture until the system has reached a temperature that favours specific primer annealing. As a result hot start PCR can greatly improve specificity, sensitivity and yield in a PCR.<br />
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===Effective Additives===<br />
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The most simple additive in our PCR is DMSO(>=99.9% purity) at a concentration of 5%(V/V). It works well in most 'problematic' PCR, however, when DMSO fails in some case, we have to turn to a magic mixed additive (2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA) for help. This excellent additive has solved several PCR where even 5% DMSO fails. <br />
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In certain PCR there is no yield without this additives, for example the ones by which we amplify bphA1A2A3A4 and bphBC.<br />
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==== Explanation ====<br />
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One major factor limiting the output of PCR routines is that a number of DNA sequences are poorly or not amplifiable under standard reaction conditions, either because of their high GC-content or/and their instrict propertoties to form secondary structures. This protocol is intended for GC-rich DNA sequences. This is a concentration dependent combination of betaine, dithiothreitol, and dimethyl suloxide. According to the references the concentration ratio is: a mixture containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA. It is stable at -20℃ for at least 3 months.<br />
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== References for special protocol: ==<br />
1.Simplified hot start PCR, David E. Birch et al. Nature 381:445-446 (1996)<br />
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2.An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose, S.Kaijalainen et al. Nucleic Acids Research 21:2959-2960 (1993)<br />
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3.Effect of DMSO on PCR of Porphyra yezoensis(Rhodophyta)gene, Yukihiro Kitade et al. Journal of Applied Phycology 15:555-557 (2003)<br />
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4.An efficient and economic enhancer mix for PCR, Markus Ralser et al. Biochemical and Biophysical Research Communications 347:747–751 (2006)<br />
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== Design Details ==<br />
Design details<br />
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[[Image:p1.jpg]]<br />
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Enhance the degradation efficiency & detect PCBs<br />
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1. Switch on the degradation pathway<br />
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We add bphR1 upstream the pathway and bphR2 downstream. bphR2 could sense the presence of PCBs and bphR1 could sense HOPDA, the third step product. These two regulators could switch on the degradation pathway. <br />
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In the absence of PCBs, small amounts of BphR2 protein bind to the bphR2 operator to re press bphR2 transcription (auto-repression). Under this condition, the levels of transcription of bph genes are very low. In the presence of PCBs, the BphR2 protein binds to the operators of bphR1 and bphA1A2A3A4BC and activates their transcription; thus high levels of HOPDA (the ring meta-cleavage compound) are produced from PCBs. In the presence of HOPDA, the transcription of bphR1 is further promoted, and its product BphR1 activates the transcription of bphR1 itself and downstream pathway [1,2].<br />
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[[Image:scheme1.jpg]] <br />
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2. Solve the bottleneck in PCBs degradation pathway<br />
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BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase), the third enzyme of the bph pathway, is of particular significance because it is incapable o f catalyzing the ring cleavage step and is subject to various types of inhabitation, as well as suicide inactivation. According to the recent research, ortho-chlorinated PCB metabolites (DHBs) are potent and physiologically significant inhibitors of BphC, so we design a feedback activation pathway using small RNA to increase the BphC transcription and expression under a 2, 3-DHBP and 4-CB inducible promoter Ppcb [3]. As 2, 3-DHBP is the second metabolite in the pathway, 2, 3-DHBP at a concentration more than 0.1mM will induce the activate PpcbC and thus increase gene expression downstream. The activated threshold of PpcbC is relatively high, so this pathway will be activated when main PCBs degradation above has began. <br />
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[[Image:s2.jpg]]<br />
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3. Control on the amount of PCBs molecular that enter the cell.<br />
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As dihydrodiols (first step product) and dihydroxybiphenyls (DHBP, second step product) are very toxic to bacterial even after short incubation time, we design a feedback repression pathway use sRNA components— sodB and rhyB. Part of sodB was fusion with bphA and biosurfactant pathway and will be repressed by rhyB[4] which is under the control the promoter Ppcb. As only when rhyB molecules is much more than sodB fusion mRNA, it will play a role of repression, only the high concentration of DHBP will arise this reaction. This design will control the amount of PCBs molecular that enter through the membrane. <br />
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[[Image:s3.jpg]]<br />
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4. System guarantee<br />
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In our design T7 amplification system is used to amplify the signal. T7 polymerase is added downstream the main degradation pathway. When T7 polymerase is translated, it will activate the T7 promoter and downstream reporter gene, YFP. Then the signal could be detected. This system could be used to test the function of the degradation pathway.<br />
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[[Image:p4.jpg]]<br />
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Reference: <br />
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[1] Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features. Kensuke Furukawa and Hidehiko Fujihara, Journal of bioscience and bioengineering, 2008, Vol. 105, No. 5:433–449,<br />
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[2] Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls. Seah S. Y., Labbe, G., Kaschabek, S. R., Reifenrath, F.,<br />
Reineke, F., and Eltis, L. D., J. Bacteriol., 2001, 183:1511–1516.<br />
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[3] Construction of transformant reporters carrying fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic pollutants. Sang-ho Park,Young Lee, Jong-Chan Chae and Chi-Kyung Kim. Environmental Monitoring and Assessment, 2004, 92:241–251.<br />
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[4] Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery. Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard, Yongchun Tang. Biotechnology and Bioengineering, 2007, Vol. 98, No. 4.<br />
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[5] ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls. Jessika Feliciano, Shifen Xu, Xiyuan Guan, Hans-Joachim Lehmle , Leonidas G. Bachas, Sylvia Daunert. Anal Bioanal Chem, 2006, 385: 807–813<br />
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== Results ==<br />
1.<br />
We have made great efforts on refining experiments protocols and shared several protocols that proved to be effecient. Our results show that you will have successful experiments if these protocols were used.<br />
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2. Plasmids constructed<br />
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We have submitted 6 standard parts including dxnA, dbfB, redA2,dxnB, bphB, bphC, bphD. These are genes coding critical enzymens in the degradation pathway of dioxin and PCBs.<br />
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3. A functional part <br />
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Promoter PpcbC and FYP was fused into a plasmids using pZE12 as the vector.</div>Sally730http://2008.igem.org/Beijing_Normal/13_October_2008Beijing Normal/13 October 20082008-10-28T12:27:43Z<p>Sally730: New page: We were told that we had to wait until the equipment is available.</p>
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<div>We were told that we had to wait until the equipment is available.</div>Sally730http://2008.igem.org/Beijing_Normal/12_October_2008Beijing Normal/12 October 20082008-10-28T12:26:35Z<p>Sally730: New page: M9 media was prepared. We plan to assay the function of promoter PpcbC.</p>
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<div>M9 media was prepared. We plan to assay the function of promoter PpcbC.</div>Sally730