Difference between revisions of "Part:BBa K902065"

Latest revision as of 16:10, 25 September 2015

Rhamnose inducible, glucose repressible promoter (pRha)

The P_rha promoter is two promoters in one. In its native E. coli, the 5' end of the promoter serves to express the rhaR and rhaS control genes. The 3' end of the promoter expresses the RhaBAD rhamnose metabolic genes.

In rhamnose, the following occurs: RhaR transcription factor is activated by rhamnose to upregulate rhaR and rhaS. RhaS in turn up-regulates the rhaBAD operon. These functions are dependent on the binding of the catabolite receptor protein (CRP) cAMP complex to the promoter. Additionally, function of RhaS is improved in the presence of rhamnose (Egan & Schleif, 1993). Note that this activation necessarily depends on CRP-cAMP (cAMP receptor protein) complex being bound to the promoter

The glucose response: The promoter is subject to repression via global catabolite repression. With glucose present, cAMP levels are low and CRP is unable to bind to the promoter. RhaS is unable to activate transcription of the rhaBAD side of the promoter and it is thus repressed by glucose.

The iGEM Calgary 2012 used the rhaBAD side of the promoter to control expression of our kill system. We selected this promoter since it is repressed by glucose and activated by rhamnose. We designed our system to constituitively express rhaS so that it is not dependent on rhamnose to be activated. Please see the design details on our [http://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation#rhamnose Wiki]. Also, we tested the promoter sans modified expression of rhaR and rhaS with GFP and S7 micrococcal nuclease.

The iGEM Harvard 2015 Team used the rhamnose promoter in our fimH plasmid (BBa_K1850000) with the intention of controlling the amount of FimH produced. Prior research indicates that there is some question as to whether rhamnose is actually titratable. That is, when a population of cells containing plasmids with the rhamnose promoter is induced, the effect that the inducer has on the population appears to be increasing linearly with the amount of inducer. But evidence in the literature suggests that at the single cell level, each cell is either completely induced or completely uninduced. Thus while rhamnose appears to be titratable on the population level, it is possible that there is no expression over some subset of cells and 100% expression over another. We wanted to see if this was the case for BBa_K902065. We found an existing iGEM part that contained BBa_K902065 and expressed GFP. Using this part, we induced with varying concentrations of rhamnose. We performed flow cytometry to measure the fluorescence of individual cells. Our results show the distribution of the fluorescence. Our data below shows that when the concentration of inducer is 0.01%, part of the population shows the same fluorescence as the control population with no inducer added. This result confirms that the that the rhamnose promoter is bimodal; that is, each cell is turned either completely off or completely on when inducer is added. This result is interesting for our project because it shows that we cannot control the level of fimH production of each cell, but only that of the cell population as a whole.

Sequence and Features