Difference between revisions of "Part:BBa K902066"

Revision as of 17:22, 28 October 2012

pRha-B0034-K082003

This component uses the rhamnose promoter (P_rha) to control GFP with an LVA tag. We used this circuit to characterize how the rhamnose promoter is affected by rhamnose, glucose, and lack of these sugars. Please view the [http://2012.igem.org/Team:Calgary/Notebook/Protocols/Prha_Characterization protocol] which we used to conduct this experiment. On our [http://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation#rhamnose Wiki], we discuss how we implemented this expression platform for control of our kill switch.

Top ten E coli expressing GFP with P_rha. Figure one: Fluorescence output was measured in response to different concentrations of glucose, rhamnose, and neither of these sugars. Overnight cultures of E. coli with K902066 were grown in LB broth. Samples were spun down, washed and resuspended in minimal media containing different sugars, and incubated at 37^oC in a 96 well plate. The 0.2% rhamnose and 0.5% rhamnose showed induction of GFP whereas cells exposed to glucose minimal similar to the sample lacking either sugar.

Figure one indicates that not only can we induce P_rha with rhamnose, but that the promoter is quite tightly regulated with minimal leaky expression without the rhamnose inducer. This makes it an excellent candidate for a killswitch construct, as it would allow for very little leaky expression of any kill genes when not wanted and an easy way of inducing the kill gene expression. We later tested this promoter out with an S7 micrococcal nuclease. This data can be found here

Sequence and Features