IGEM:IMPERIAL/2008/Projects/motility/Alternatives

Though the blue light sensing sytem in B. subtilis can be used to detect light, it triggers the stress response which leads to sporulation. As we do not want B. subtilis to sporulate it may be neccesary to prevent this by preventing production of σB by various methods.

if this is done, an alternative light sensing pathway may be required...

AppA-PpsR


The Blue light sensor we decide to utilise is from the 'purple' anoxytrophic bacterium Rhodobacter sphaeroides. In particular, this class of bacteria tend to live in the sea, where blue light penetrates deepest and so it is beneficial for the organism to sense blue light to determine if there is enough light for effective photosynthesis. For this purpose, R.sphaeroides uses a BLUF family protein; AppA. BLUF family proteins all contain a BLUF domain which is capable of binding a flavin molecule (FMD, FAD or Riboflavin molecule). They also have a conserved glutamine residue capable of supplying a proton to the co-factor molecule upon absorption of a the energy of a photon resulting in a conformational change as AppA binds an oxidised flavin. Under dark conditions the AppA flavin remains reduced and AppA can bind the repressor protein PpsR to form a complex (AppA-PpsR2)which is the repressor of the promoters for the Photosystem genes eg. puc operon, effectively making AppA an anti-repressor. When oxidised however, Appa releases PpsR which forms a tetramer complex and binds the promoter of puc operon (and a few others)at a pair of palindromic DNA sequences. These sequences (TGTN12ACA) contain a large 12 nucleotide section that can be any base, this will make it easier to incorporate into a new promoter domain, as all the photosystem regulatory promoters all have only a single negative repressor and the aided toogle switch mechanism we are using ideally has two independent repressor elements.

The AppA/PpsR two component signalling system was chosen for its simplicity (only 2 proteins, Score!) and for this simple and easy to implant (a quick bit of site-directed mutagenesis) promoter operator.

It is worth noting however that the homologues of AppA and PpsR in other anoxytrophic purple bacteria also integrate redox signals through these proteins and so the redox state of any host we put this system in may affect the repression strength (though blue light appears to be the main factor) and this will make thorough testing and modelling a necessity. The Finished Device (Diagram):