"
Team:Evry/Biology/Sensors
From 2014.igem.org
(Difference between revisions)
| Line 62: | Line 62: | ||
<div align="justify"> | <div align="justify"> | ||
<ul> | <ul> | ||
| + | <li><u><b><i>A system of detection and degradation of polychlorinated biphenyls in Pseudomonas pseudoalcaligenes KF707</u></b></i> | ||
| + | <br/><br/> | ||
Pseudomonas pseudoalcaligenes KF707 is one of the strain which are able to degrade polychlorinated biphenyls (PCBs). This strain can grow on biphenyl and salicylate as a sole source of carbon. The bacteria contains a biphenyl-catabolic (bph) gene cluster (bphR1A1A2-(orf3)-bphA3A4BCX0X1X2X3D) which degrade compounds (figure A). | Pseudomonas pseudoalcaligenes KF707 is one of the strain which are able to degrade polychlorinated biphenyls (PCBs). This strain can grow on biphenyl and salicylate as a sole source of carbon. The bacteria contains a biphenyl-catabolic (bph) gene cluster (bphR1A1A2-(orf3)-bphA3A4BCX0X1X2X3D) which degrade compounds (figure A). | ||
<br/><br/> | <br/><br/> | ||
| Line 76: | Line 78: | ||
</a> | </a> | ||
</div> | </div> | ||
| - | <center><u>Figure A: Organization of the <i>bph</i> gene cluster from Pseudomonas pseudoalcaligenes which is implied in degradation of PCBs (K. Furukawa and H. Fujihara, 2008)</u> </center> | + | <center><u><b>Figure A: Organization of the <i>bph</i> gene cluster from Pseudomonas pseudoalcaligenes which is implied in degradation of PCBs (K. Furukawa and H. Fujihara, 2008)</u></b> </center> |
</div> | </div> | ||
</div> | </div> | ||
| Line 95: | Line 97: | ||
</a> | </a> | ||
</div> | </div> | ||
| - | <center><u>Figure B: Cross-regulation of bph gene cluster by bphR1 and bphR2 in Pseudomonas pseudoalcaligenes KF707 (K. Furukawa and H. Fujihara, 2008)</u></center> | + | <center><u><b>Figure B: Cross-regulation of bph gene cluster by bphR1 and bphR2 in Pseudomonas pseudoalcaligenes KF707 (K. Furukawa and H. Fujihara, 2008)</u></b></center> |
</div> | </div> | ||
</div> | </div> | ||
| - | |||
</div> | </div> | ||
| - | <br/> | + | </div> |
| - | <br/>In 2013, Saclays' team wanted to construct a <a href="https://2013.igem.org/Team:Paris_Saclay/Project">biosensor for PCBs</a>, a project that failed (figure C). So our first aim was to do their construction | + | <br/><li><u><b><i>2013 Saclays' project : </u></b></i> |
| + | <br/><br/> | ||
| + | In 2013, Saclays' team wanted to construct a <a href="https://2013.igem.org/Team:Paris_Saclay/Project">biosensor for PCBs</a>, a project that failed (figure C). So our first aim was to do their construction and then to optimize it and to characterize it. | ||
<br/><div class="center"> | <br/><div class="center"> | ||
<div class="thumb tnone"> | <div class="thumb tnone"> | ||
| Line 114: | Line 117: | ||
</a> | </a> | ||
</div> | </div> | ||
| - | <center>Figure C: 2013 Saclay's project: construction of a PCBs biosensor</center> | + | <center><u><b>Figure C: 2013 Saclay's project: construction of a PCBs biosensor</u></b></center> |
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| - | + | <li><u><b><i>How function our biosensor ?</i></b></u><br/><br/> | |
| - | + | Our construction is composed by a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23114">(BBa_J23114)</a>, a RBS <a href="http://parts.igem.org/Part:BBa_B0034">(BBa_B0034)</a>, bphR2 gene <a href="http://parts.igem.org/Part:BBa_K1413021">(BBa_K1413021)</a>, which has been mutated because of a pstI site in its sequence, bphR1 promoter region <a href="http://parts.igem.org/Part:BBa_K1155001">(BBa_K1155001)</a>, received from Saclay’s team, RFP <a href="http://parts.igem.org/Part:BBa_E1010">(BBa_E1010)</a> and a terminator <a href="http://parts.igem.org/Part:BBa_B0015">(BBa_B0015)</a> (figure D). Instead to use a system of detection with lacZ, we have opted for a fluorescence system with RFP. | |
| - | + | <br/><br/> | |
| - | <br/><br/> | + | <div align="center"><img src="https://static.igem.org/mediawiki/2014/a/a6/IGEM_Evry2014_Sensor_part.png" width="700px"; alt="image not found"/></div><br/><br/> |
| - | + | <u> In absence of PCBs : </u>bphR2 <a href="http://parts.igem.org/Part:BBa_K1413021">(BBa_K1413021)</a> is bound to bphR1 promoter which activate the transcription of RFP but in very low expression. | |
| - | + | <br/><u> In presence of PCBs : </u>When compound diffuses into the media, it binds to bphr2 protein which undergoes a conformational change that permits to activate more stronger bphR1 promoter and increase the transcription of RFP. | |
<br/> | <br/> | ||
<br/> | <br/> | ||
| - | + | <li><u><b><i>Improvement</i></b></u> | |
| - | + | <br/> | |
| - | + | <br/> | |
| - | + | In order to improve their construction, we have made some modifications. In fact, we have used to add RBS B0034 to increase the level of transcription. After bphR2 gene, we have put a terminator to prevent leakage of constitutive promoter. In their sequence of bphR2 <a href="http://parts.igem.org/Part:BBa_K1155009">(BBa_K1155009)</a>, there is a pstI site so this gene has been mutated by keeping the same codon <a href="http://parts.igem.org/Part:BBa_K1413021">(BBa_K1413021)</a>. | |
| - | + | <br/> | |
| - | + | <br/> | |
| - | + | <li><u><b><i>Construction of our biosensor in E. coli DH5a : </u></b></i> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
<br/> | <br/> | ||
| - | + | <br/> | |
| + | Every parts come from the registry except bphr2 gene. Knowing that there is a pstI site in its sequence, bphR2 gene has been synthetized without this site to allow to do the construction at biobrick format. | ||
| + | <br/> | ||
| + | <br/> | ||
| + | <b>Golden gate : </b> | ||
| + | <br/> We wanted to assemble all the parts by using golden gate so in one shot. For that, we have followed this processus: | ||
| + | <ul> | ||
| + | <li>Transformation of all the parts in DH5a by heat shock | ||
| + | <li>Miniprep | ||
| + | <li>PCR with primers designed especially for the golden gate. Theses primers have been designed so as to have a bsaI site. | ||
| + | <li>PCR clean-up | ||
| + | <li>Launching of golden gate program (cf notebook) | ||
| + | <li> Transformation in DH5a | ||
| + | </ul> | ||
| + | <br/>=> Several tests have been done without success. | ||
| + | <br/><br/> | ||
| + | <b>Sequential digestions : </b> | ||
| + | <br/><br/> | ||
| + | Each part contains biobrick prefix and biobrick suffix so we have decide to do a digestion-ligation-transformation protocol (figure F) | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
</ul><br/> | </ul><br/> | ||
| + | |||
Revision as of 22:01, 17 October 2014
Biology - Classic & RNAseq-based sensors
Phenol biosensor :
- Biosensor construction
- Dmpr, a phenol dependant signal transducer
Presentation
DmpR is a member of NtrC protein family. NtrC-type regulators activate RNAP containing the alternative sigma factor 54. The s54-RNAP holoenzyme forms a stable complex with -12 and -24 promoters but is unable to start transcription without further activation NtrC protein family strongly stimulate the polymerase complex. They bind to DNA regions more than 100bp upstream from the s54-RNAP binding site (UAS). Interaction between the regulatory protein and s54-RNAP is facilitated by a bend of DNA.
Structure
DmpR is a 563 amino acid long protein. Although no direct structural information have been described (e.g protein purification), comparisons with other member of NtrC family and genetic experiments have brought some insight on its structure. Four domains are classically described for members of NtrC family :
A-domain (211 amino acid long) involved in direct interaction with effector. One inducer binding site is present per monomer, which was demonstrated for DmpR and which could be pinpointed to a subregion between amino acid residues 107 and 186
B-domain is a linker between A and C domain.
C-domain being the most highly conserved region among the family members, is involved in ATP binding and hydrolysis and in s54-RNAP interaction
D-domain for Dna binding with HTH motif (helix turn helix).
Effectors dependant activation
DmpR-like activators require a chemical effector and ATP as the cofactor. The effector is usually the primary substrate of the target pathway or a compound related to this. Phenol and its derivative are typical effector for DmpRMechanism
• 1. Binding with effector : The key control event is a direct interaction of aromatic effectors (phenol) with A-domain, which leads to the expression of its otherwise repressed C-domain mediated ATPase activity (Shingler and Pavel, 1995). B domain act as a linker between A and C domains and is necessary for C domain derepression in presence of effector.
PCBs biosensor :
- A system of detection and degradation of polychlorinated biphenyls in Pseudomonas pseudoalcaligenes KF707
Pseudomonas pseudoalcaligenes KF707 is one of the strain which are able to degrade polychlorinated biphenyls (PCBs). This strain can grow on biphenyl and salicylate as a sole source of carbon. The bacteria contains a biphenyl-catabolic (bph) gene cluster (bphR1A1A2-(orf3)-bphA3A4BCX0X1X2X3D) which degrade compounds (figure A).
Figure A: Organization of the bph gene cluster from Pseudomonas pseudoalcaligenes which is implied in degradation of PCBs (K. Furukawa and H. Fujihara, 2008)
bphR1 is a positive regulator for bphR1 protein, which belongs to the GntR family, and for bphX0X1X2X3D. It's implied in the degradation of PCBs. Watanabe et al showed that there is an other regulatory protein, bphr2, which is involved in the positive regulation of bphA1A2A3A4BC genes. In the absence of biphenyl, small amounts of bphR2 protein binds to bphR2 operator to repress bphR2 transcription (autorepression) and activate bphR1 weakly. When there is biphenyl in the media, bphR2 protein binds to bphR1 and bphA1A2A3A4BC operators to activate strongly their transcription. This allows to initiate the degradation of biphenyl. (figure B).
Figure B: Cross-regulation of bph gene cluster by bphR1 and bphR2 in Pseudomonas pseudoalcaligenes KF707 (K. Furukawa and H. Fujihara, 2008) - 2013 Saclays' project :
In 2013, Saclays' team wanted to construct a biosensor for PCBs, a project that failed (figure C). So our first aim was to do their construction and then to optimize it and to characterize it.
Figure C: 2013 Saclay's project: construction of a PCBs biosensor - How function our biosensor ?
Our construction is composed by a constitutive promoter (BBa_J23114), a RBS (BBa_B0034), bphR2 gene (BBa_K1413021), which has been mutated because of a pstI site in its sequence, bphR1 promoter region (BBa_K1155001), received from Saclay’s team, RFP (BBa_E1010) and a terminator (BBa_B0015) (figure D). Instead to use a system of detection with lacZ, we have opted for a fluorescence system with RFP.
In absence of PCBs : bphR2 (BBa_K1413021) is bound to bphR1 promoter which activate the transcription of RFP but in very low expression.
In presence of PCBs : When compound diffuses into the media, it binds to bphr2 protein which undergoes a conformational change that permits to activate more stronger bphR1 promoter and increase the transcription of RFP.
- Improvement
In order to improve their construction, we have made some modifications. In fact, we have used to add RBS B0034 to increase the level of transcription. After bphR2 gene, we have put a terminator to prevent leakage of constitutive promoter. In their sequence of bphR2 (BBa_K1155009), there is a pstI site so this gene has been mutated by keeping the same codon (BBa_K1413021).
- Construction of our biosensor in E. coli DH5a :
Every parts come from the registry except bphr2 gene. Knowing that there is a pstI site in its sequence, bphR2 gene has been synthetized without this site to allow to do the construction at biobrick format.
Golden gate :
We wanted to assemble all the parts by using golden gate so in one shot. For that, we have followed this processus:- Transformation of all the parts in DH5a by heat shock
- Miniprep
- PCR with primers designed especially for the golden gate. Theses primers have been designed so as to have a bsaI site.
- PCR clean-up
- Launching of golden gate program (cf notebook)
- Transformation in DH5a
=> Several tests have been done without success.
Sequential digestions :
Each part contains biobrick prefix and biobrick suffix so we have decide to do a digestion-ligation-transformation protocol (figure F)
- Dmpr, a phenol dependant signal transducer
Phenol and its derivative are of major concern since their accumulation in the environment, as a result of
intensive human activity, cause toxicity for both flora and fauna [1] [2].In the context of bioremediation and preservation of water we designed a biosensor of phenol that allows us to measure the concentration of phenol in a given marine environment.
This phenol biosensor rely on both signal transducing component, DmpR and inducible fluorescence emitting component based on the Green Fluorescent Protein (GFP). Both elements are assembled in a single plasmid and allow bacteria to respond to the presence of phenol by emitting fluorescence.
