IGEM:IMPERIAL/2008/Bioprinter/Subteam 2/ytva

Promoter
The promoter pctc from is a σB regulated promoter that has been extensively studied. In fact it has been used to design reporting vectors for measuring σB activity, this is achieved by a pctc-lacZ construct. Studies have shown that we require ~50bp upstream of the start site to allow functional transcription by σB pr1. The sequence of this stretch is shown below, along with the contact sites of σB and the start site.

YtvA Protein


Previous studies have shown that if the YtvA protein is not overexpressed within B.subtilis then the response from a pctc reporter construct is low. Only when overexpression is high is there a significant increase in pctc expression in the prescence of blue light. The levels of light that the B.subtilis was exposed to was 30 microeinsteins m-2s-1. The relative response time of the pctc promoter has not been tested, a previous paper has shown that the expression of the YtvA protein and induction of the pctc promoter takes ~60 minutes. A study of the recovery time of YtzA has shown that the initial sensing of blue light very quick, but the recovery of the chromophore takes around 45 minutes Recovery. Here is a link to the sequence of the YtvA protein: Sequence

Pathways
The model of the σB signal transduction network is shown on the right. Stress signals are conveyed to σB via a network that functions by a partner-switching mechanism. When B.Subtilis is not under stress, RsbV is phosphorylated and cannot interact with RsbW anti-σ which binds σB in an inactive complex. Under stress, RsbV-P is dephosphorylated, allowing it to bind to RsbW, stimulating the release of σB. Under environmental stress, RsbT phosphorylates RsbRA and RsbS, releasing RsbT to activate the RsbU phosphatase. The RsbX feedback phosphatase returns the system to its original unstressed state. SigB

Reactions Constants

 * The kinetics of the dark recovery reaction for YtvA-LOV is slower than for full-length YtvA, with τrec = 3900 and 2600 s at 25°C. YtvA
 * YtvA and YtvA-LOV exhibited the same rate constant of the photoadduct formation, 4.0×10^2 s^-1, whereas the rate constant of dark reversion for YtvA-LOV was 2.7×fold faster than that for YtvA; 4.8×10^4 s^-1 and 13.0×10^4 s^-1 for YtvA and YtvA-LOV, respectively. YtvA2