Part:BBa_K2969020

CI434ts-TEVsite

CI434ts is a thermo-sensitive mutation type of CI434 transcription factor. It is created through an evolutionary strategy which use temperature as a screening condition. ‘ts’ means thermo-sensitive. It is a heat-inducible transcription factor. When the temperature is high, it will function and repress its promoter to inhibit the process of transcription.

Characterization

In 2019, UCAS-China made several improvements to CI434, BBa_C0056 by constructing a new part, CI434-TEVsite, BBa_K2969020. Firstly, we inserted the TEV protease recognition sequence between N-terminal and C-terminal of CI434 so that it can be cleaved by TEV and then lose activity, which can regulate gene expression at both transcriptional and proteolytic levels. As shown in Figure 1 and Figure 2, we can see that CI434-TEVsite and original TEV have little difference. However, when there exists TEV protease, CI434-TEVsite can lose its activity so that the genes expression under CI434-TEVsite can be activated significantly.

Figure 1:The effect of CI434 and CI434-TEVsite at different temperatures

Figure 2:The cleavage efficiency of TEV to CI434-TEVsite induced by a series of concentrations of IPTG

Besides, we also developed CI434ts-TEVsite through an evolutionary strategy which use temperature as a screening condition. CI434ts is a heat-inducible ON-transcription factor which is a thermo-sensitive mutation type of CI434 transcription factor, which can regulate gene expression at different temperatures. As shown in Figure 3, when the temperature is high, CI434ts will function and repress its promoter to inhibit the process of under-stream genes transcription more tightly than original CI434.

Figure 3:The effect of CI434 and CI434ts at different temperatures

Therefore, our part, CI434ts-TEVsite have both functions and can be applied in many ways.

Reference

Zheng, Y., Meng, F., Zhu, Z., Wei, W., Sun, Z., Chen, J., . . . Chen, G.-Q. (2019). A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts. Nucleic Acids Research. doi:10.1093/nar/gkz785

Contribution by UCAS-China 2020

We got the quantitative measurement of the cI434 variant and its controls: cI434wt is the wild-type cI434 repressor with an inserted TEV cleavage site; cI434ts is a selected cold-inactivated cI434 repressor. The cold-inactivated cI434 variant (cI434ts) exhibited approximately 3-fold induction when the temperature was shifted from 37 to 25°C.

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