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Part:BBa_K1461207:Experience

Designed by: Takefumi Yoshikawa   Group: iGEM14_UT-Tokyo   (2014-09-26)


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[http://2014.igem.org/Team:UT-Tokyo/Counter/Project?page=Result-block&contents=Result-1 iGEM UT-Tokyo 2014]

T--UT-Tokyo--BBa K1461207--1.png
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[http://2016.igem.org/Team:UT-Tokyo/Project#result iGEM UT-Tokyo 2016]

When its functionality was tested previously, by iGEM-UT Tokyo 2014(above), the results suggested that the anti sigma 11 was not able to repress the activity of ECF 11. It was hypothesised that anti sigma11 not functioning due to “ the sigma factors [were] expressed more than the anti-sigma factors could repress their activation of the corresponding promoters.” Thus, we attempted a new characterisation this time ligating anti 11 on a high copy plasmid and ECF sigma11 on a low copy plasmid in order to increase the level of anti 11 relative to sigma11.

Functionality assay of anti 11 by increasing concentration of anti11 relative to sigma11. Instead of generating sigma11 on backbone pSB1C3 as done in by iGEM UT-Tokyo 2014, sigma 11 was generated on backbone pSB3K3 and anti11 in pSB1C3 as an attempted to increase the level of anti11 relative to sigma11. The measurement shows that there is a significant level of difference between the anti sigma 11(-) construct and antisigma 11 (+). However, the difference between the complete OFF state (without sigma) and with anti 11 state is also very significant. Data represents an average of 3 samples with error bars representing the standard deviation.

We have experimentally validated that anti sigma11 can function to inhibit the activity of sigma11 as the fluorescence output from the sigma 11 promoter was significantly reduced in presence of anti11. Nevertheless, it should be noted that the anti sigma11 is not strong enough to function as a “switch” that can turn the promoter back to the state when there is no ECF sigma11 present.

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