Difference between revisions of "Part:BBa K395301:Experience"
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[[Image:Tokyotech_ompc_graph.jpg|thumb|left|400px|Figure 1. The induction of new'' OmpC'' series in high osmolarity medium ]] | [[Image:Tokyotech_ompc_graph.jpg|thumb|left|400px|Figure 1. The induction of new'' OmpC'' series in high osmolarity medium ]] | ||
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+ | PompC(C)-GFP, PompC(CB)-GFP PompC(CS1)-GFP and PompC(WT)-GFP on pSB3K3 was introduced into E. coli strain MG1655. A strain containing pLacI<sup>q<sup>-GFP, plasmid (BBa_J54202), a constitutive GFP expressive promoter, and promoterless GFP reporter plasmid (BBa_J54103) was 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 see more about PompC series | ||
+ | After 4 hours of sucrose induction, 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. | ||
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===Applications of BBa_K395301=== | ===Applications of BBa_K395301=== | ||
Revision as of 11:36, 26 October 2010
PompC(C)-GFP, PompC(CB)-GFP PompC(CS1)-GFP and PompC(WT)-GFP on pSB3K3 was introduced into E. coli strain MG1655. A strain containing pLacIq-GFP, plasmid (BBa_J54202), a constitutive GFP expressive promoter, and promoterless GFP reporter plasmid (BBa_J54103) was 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 see more about PompC series After 4 hours of sucrose induction, 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.
Applications of BBa_K395301
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