Team:EPF-Lausanne/R-BphP

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== The BphP / PpsR system ==
 
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PpsR1 is a redox sensitive activator. It binds DNA under anaerobic conditions, and forms a tetramer via a disulfide bond. This interaction is ablated in the mutant PpsR1-C429S; meaning that we should be able to mimic an anaerobic system even under aerobic conditions.
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<font size="6" color="#007CBC"><i>R-BphP project </i></font>
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PpsR2 is a transcriptional repressor, regulated by BphP.
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This part of the project was cancelled, due to lack of time. You can read what our idea was on [https://2009.igem.org/Team:EPF-Lausanne/Brainstorming the brainstorming session]
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PpsR1 and PpsR2 bind to TGTN_12ACA possibly arranged in tandem with a 7 base spacer. The affinity for both PpsRs are around 100nM.
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BphP (or BrBphP for the Bradyrhizobium variant) is sensitive to far-red light (~770nm) and controls PpsR1.
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For more info, see [http://www.jbc.org/cgi/content/full/279/43/44407].
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Similar constituents could possibly be derived from R. Palustris as well with the following model derived from [http://www3.interscience.wiley.com/cgi-bin/fulltext/118512333/HTMLSTART]:
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[[Image:R._palustris_circuit.gif]]
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==To find==
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- Define genetic circuit <big>'''Tú'''</big>
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<br> - Look for exact wavelength for the corresponding light receptor<big><span style="color:Blue"> Basile</span></big>
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<br> - Sequences of the genes involved in the pathway (minimal genes to  => [http://www.ncbi.nlm.nih.gov/ Pubmed])
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<br>[[Media:‎ ORS278_BrBphP_genomic_seq.txt|ORS278 BrBphP genomic sequence]]
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<br>[[Media:‎ ORS278_PpsR1_genomic_seq.txt|ORS278 PpsR1 genomic sequence]]
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<br>[[Media:‎ ORS278_PpsR2_genomic_seq.txt|ORS278 PpsR2 genomic sequence]]
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<br>[[Media:‎ ORS278_PpsR1-C429S_protein_seq.txt|ORS278 PpsR2-C429S protein sequence]]
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==Implementation==
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There are two modes in which we can use the system. The minimal complement requires PpsR2 and its regulator BphP. We can generate a hybrid system that uses a well known activator or transcription or a constitutively active promoter which will be repressed via PpsR2 upon exposure to far-red light (770nm).
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It is also possible to reconstitute the entire system including PpsR1-C429S, which will serve as the activator and will be de-coupled from the oxidative state of the cell due to the ablation of the disulfide bond formation.  
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==To do==
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* contact Eric Giraud (giraud@mpl.ird.fr) and ask if he could send us the following material:
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** Bradyrhizobium ORS278
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** The following plasmids:
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*** pBAD::ppsR1
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*** pBAD::ppsR2
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*** pGEM-T::ppsR2/BrbphP
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** the PpsR1-C429S construct
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==Found==
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- Bacterium: Bradyrhizobium  can be ordered on [http://www.lgcstandards-atcc.org/LGCAdvancedCatalogueSearch/ProductDescription/tabid/1068/Default.aspx?ATCCNum=10244&Template=bacteria ATCC]
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!align="center"|[[Team:EPF-Lausanne|Home]]
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!align="center"|[[Team:EPF-Lausanne/Team|The Team]]
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!align="center"|[[Team:EPF-Lausanne/Project|The Project]]
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!align="center"|[[Team:EPF-Lausanne/Parts|Parts Submitted to the Registry]]
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!align="center"|[[Team:EPF-Lausanne/Modeling|Modeling]]
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!align="center"|[[Team:EPF-Lausanne/Notebook|Notebook]]
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!align="center"|[[Team:EPF-Lausanne/Lectures|Lectures]]
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Latest revision as of 08:07, 11 October 2009




R-BphP project



This part of the project was cancelled, due to lack of time. You can read what our idea was on the brainstorming session

Retrieved from "http://2009.igem.org/Team:EPF-Lausanne/R-BphP"