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| - | {{EPF-Lausanne09}}
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| - | <div CLASS="epfltrick">__TOC__
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| - | </div><div CLASS="epfl09">
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| - | <html><center>
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| - | <font size="6" color="#007CBC"><i>R-BphP Project</i></font>
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| - | </center></html>
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| - | <br>
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| - | ----
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| - | <br>
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| - | '''Finally this part of the project was cancelled, due to lack of time.'''
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| - | But it is still of interest to describe it, it could be useful in the future.
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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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| - | PpsR2 is a transcriptional repressor, regulated by BphP.
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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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| - | ==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 factor, 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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| - | ==Strains==
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| - | * We contacted Eric Giraud 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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| - | * We received the strains in late July, thanks the Eric Giraud's team !
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| - | ==The pathway==
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| - | - Sequences of the genes involved in the pathway (minimal genes to => [http://www.ncbi.nlm.nih.gov/ Pubmed])
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| - | [[Media: ORS278_BrBphP_genomic_seq.txt|ORS278 BrBphP genomic sequence]]
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| - | [[Media: ORS278_PpsR1_genomic_seq.txt|ORS278 PpsR1 genomic sequence]]
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| - | [[Media: ORS278_PpsR2_genomic_seq.txt|ORS278 PpsR2 genomic sequence]]
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| - | [[Media: ORS278_PpsR1-C429S_protein_seq.txt|ORS278 PpsR2-C429S protein sequence]]
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| - | [[Media: hmuO_ORS278 Brad.txt|ORS278 hmuO genomic sequence]]
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| - | [[Media: ppsR1_prot_RPalustris.txt| R.Palustris CGA009 ppsR1 protein sequence]]
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| - | [[Media: ppsR1_RPalustris.txt| R.Palustris CGA009 ppsR1 genomic sequence]]
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| - | [[Media: ppsR2_prot_RPalustris.txt| R.Palustris CGA009 ppsR2 protein sequence]]
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| - | [[Media: ppsR2_RPalustris.txt| R.Palustris CGA009 ppsR2 genomic sequence]]
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| - | [[Media: CAE001 bphP1.txt| R.Palustris CAE001 bphP1 genomic sequence]]
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| - | [[Media: CGA009 hmuO.txt| R.Palustris CGA009 hmuO genomic sequence]]
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| - | [[Image: bphp_organisation.gif|500px|thumb|center| Organization of the genes downstream the puf operon in Bradyrhizobium and Rps. palustris. BphP: bacteriophytochrome gene; hmuO: heme oxygenase gene.]]
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