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

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P''ompC(C)''-GFP, P''ompC(CB)''-GFP, P''ompC(CS1)''-GFP and P''ompC(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.  
 
P''ompC(C)''-GFP, P''ompC(CB)''-GFP, P''ompC(CS1)''-GFP and P''ompC(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.  
 
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.  
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After 4 hours of sucrose induction, transcriptional activity of P''ompC(CB)''-GFP and P''ompC(CS1)''-GFP increased 2.5 folds and 2.3 folds respectively. However, significant amount of leaky expression was found in P''ompC(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 P''ompC'' series]
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After 4 hours of sucrose induction, transcriptional activity of P''ompC(CB)''-GFP and P''ompC(CS1)''-GFP increased 2.5 folds and 2.3 folds respectively. However, significant amount of leaky expression was found in P''ompC(CS1)''-GFP without induction. In contrast, under the same conditions, we found no significant difference of GFP expression in P''ompC(C)''-GFP and P''ompC(WT)''-GFP. [http://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC ...see more about P''ompC'' series]
  
 
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Revision as of 11:59, 26 October 2010

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Applications of BBa_K395301

User Reviews

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Tokyo Tech iGEM2010

Figure 1. Induction of new OmpC series in high osmolarity medium

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. 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. [http://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC ...see more about PompC series]

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