Team:EPF-Lausanne/R-BphP

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 Tú
- Look for exact wavelength for the corresponding light receptor Basile
- 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

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 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

Found

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