Difference between revisions of "Part:BBa K395301:Experience"

Tokyo Tech iGEM2010

Figure 1. Induction of new OmpC series in high osmolarity medium at 4 hours. We characterized this promoter by attaching GFP(E0240) to the downstream and mearsuring its transcriptional activity through the GFP expression. This work is done by Thiprampai THAMAMONGOOD and Taichi NAKAMURA 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. Although, PompC(WT)-GFP also doesn't show the difference in expression of GFP at 4 hours of induction, there was slightly higher GFP expression 1.7-folds occurred in the wild type at 2 hours. We considered that the transcriptional activity of PompC(WT)-GFP increased in particular period and declined after it reached the peak of expression. This result also shows the relation to the study by the Edinburgh team iGEM 2009.[http://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC ...see more about PompC series]