Difference between revisions of "Part:BBa K902066"

Latest revision as of 19:21, 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 showed minimal fluorescence 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 minimal expression of kill genes when when activators are not present. P_rha is more tightly regulated as opposed to other expression platforms in the registry. Consider, for example, the LacI promoter which show significant leaky expression without IPTG inducer (see Figure 4C where we showed this leaky expression on our [http://2012.igem.org/Team:Calgary/Project/FRED/Reporting electrochemical reporting page]).

Note that this construct does not involve the use of either rhaR or rhaS regulatory elements. Induction of this construct was entirely dependent on levels of these regulatory proteins from the native E coli. genome. In future trials, we hope characterize P_rha by also expressing the two control proteins on the constructs. Other teams might try placing rhaS and rhaR on the opposite side of P_rha (see the P_rha page to learn of how this promoter also controls rhaR and rhaS). For the purpose of P_rha and our kill system, we wish attempt constitutive expression of rhaS so that our system is not dependent on the presence of rhamnose to activate the kill genes. More experimentation is necessary.

We later tested this promoter out with an S7 micrococcal nuclease. This data can be found here.

Sequence and Features