Difference between revisions of "Part:BBa R0082:Experience"

No review score entered. Edinburgh iGEM 2009

We tested this promoter for use in our landmine-detection system. Our plan was to use this promoter together with a computationally designed TNT receptor and the hybrid Trz signal transduction protein, which has the EnvZ kinase domain, hence phosphorylates OmpR, activating this promoter, in the presence of TNT. To check that this promoter was working correctly, we attached it to GFP and assayed its activity in E. coli TOP10 cells as described on the Registry Measurement Page. (We also made a lacZ reporter version, BBa_K216010, so that we could do Miller assays as a backup, but at the time of writing we have not obtained good quantitative results from this). To induce the promoter, we used procaine, which activates the EnvZ kinase. Our results indicated that the promoter is activated by increasing levels of procaine from 0 to 15 mM, but there is very high basal activity under the conditions used, even in the absence of procaine. We also made an envZ mutant host strain using the KEIO collection of E. coli mutants, but at the time of writing, we have not yet repeated the tests using this host.

Tokyo Tech iGEM2010

Figure 1. Induction of new OmpC series in high osmolarity medium at 4 hours Tokyo Tech iGEM2010

In order to characterize PompC(C) BBa_395301, PompC(CB) BBa395302 and PompC(CS1) BBa_395303, each promoter was attached to GFP and its transcriptional activity was measured through the GFP expression.

PompC(C)-GFPBBa_395304 , PompC(CB)-GFP BBa_395305, PompC(CS1)-GFP BBa_395306and PompC(WT)-GFPBBa_395307 on pSB3K3 was introduced into E. coli strain MG1655. A strain containing placI^q-GFP, plasmid (BBa_J54202), a constitutive GFP expressive promoter and promoterless GFP reporter plasmid (BBa_J54103) were used as a positive control and negative control respectively.

Overnight cultures of reporter strains grown at 37 °C in Medium A containing appropriated antibiotics were diluted at least 1:100 in the medium and incubated at 37 °C as fresh cultures. After their OD₅₉₀ reached 0.2, the fresh culture was diluted 1 : 3 into 2 ml of pre-warmed medium A. For high osmolarity conditions, the cultures were diluted with sucrose supplemented medium to the final concentration of 15% (wt/vol). After 4 hours of induction, fluorescence intensity was measured with fluorometer.

After 4 hours of high osmolarity induction by sucrose, transcriptional activity of PompC(CB)-GFP and PompC(CS1)-GFP increased 2.5 folds and 2.3 folds respectively. However, significant amount of leaky expression was found in PompC(CS1)-GFP without induction. In contrast, under the same conditions, we found no significant difference of GFP expression in PompC(C)-GFP and PompC(WT)-GFP. [http://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC ...see more about PompC series]