Team:EPF-Lausanne/R-BphP

Finally this part of the project was cancelled, due to lack of time.

The BphP / PpsR system

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.

PpsR2 is a transcriptional repressor, regulated by BphP.

PpsR1 and PpsR2 bind to TGTN_12ACA possibly arranged in tandem with a 7 base spacer. The affinity for both PpsRs are around 100nM.

BphP (or BrBphP for the Bradyrhizobium variant) is sensitive to far-red light (~770nm) and controls PpsR1.

For more info, see [http://www.jbc.org/cgi/content/full/279/43/44407].

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]:

To find

- Define genetic circuit

- Look for exact wavelength for the corresponding light receptor

- Sequences of the genes involved in the pathway (minimal genes to => [http://www.ncbi.nlm.nih.gov/ Pubmed])

ORS278 BrBphP genomic sequence

ORS278 PpsR1 genomic sequence

ORS278 PpsR2 genomic sequence

ORS278 PpsR2-C429S protein sequence

ORS278 hmuO genomic sequence

R.Palustris CGA009 ppsR1 protein sequence

R.Palustris CGA009 ppsR1 genomic sequence

R.Palustris CGA009 ppsR2 protein sequence

R.Palustris CGA009 ppsR2 genomic sequence

R.Palustris CAE001 bphP1 genomic sequence

R.Palustris CGA009 hmuO genomic sequence

Organization of the genes downstream the puf operon in Bradyrhizobium and Rps. palustris. BphP: bacteriophytochrome gene; hmuO: heme oxygenase gene.

Implementation

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).

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.

To do

• contact Eric Giraud (giraud@mpl.ird.fr) and ask if he could send us the following material:
  • Bradyrhizobium ORS278
  • The following plasmids:
    • pBAD::ppsR1
    • pBAD::ppsR2
    • pGEM-T::ppsR2/BrbphP
  • the PpsR1-C429S construct
• We received the strains in late July, thanks the Eric Giraud's team !

Found

- Bacterium: Bradyrhizobium can be ordered on [http://www.lgcstandards-atcc.org/LGCAdvancedCatalogueSearch/ProductDescription/tabid/1068/Default.aspx?ATCCNum=10244&Template=bacteria ATCC]