Difference between revisions of "Part:BBa K2150031"

Revision as of 14:54, 21 October 2019

T7 promoter without RBS

This part is the T7 promoter without RBS. It has been reported that T7 promoter doesn't work with E.coli RNA polymerase, but only with T7 RNA polymerase(T7RNAP). However, we observed leakage of it in the absence of T7RNAP.

Improvement: Jilin_China 2019

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1. Design

T7 promoter is one of the most common promoters. But the intensity of the wild type T7 promoter often can’t meet our demand, so we mutated it and obtained an improved T7 promoter. According to previous studies, we selected the mutation site and constructed three mutated promoters (BBa_K2150012, BBa_K2150013, BBa_K2150014）based on the wild-type T7 promoter (BBa_K2150031) into the measurement vector (BBa_K3078100).

2. Measurement

The promoter activity of wild-type and mutant T7 were measured through fluorescence intensity and normalized by OD₆₀₀. BBa_K2150031 and the mutated T7 promoter lack of T7 RNA polymerase, so we added IPTG to induce the expression of T7 RNA polymerase downstream of promoter lacUV5 of on the BL21(DE3) genome, as shown in Figure 2.

It can be seen from the figure that the promoter strength of mutant is higher than wild type.

3. Conclusion

In conclusion, we constructed wild-type and mutanted T7 promoters into the measurement vector (BBa_K3078100) by Golden Gate method, and confirmed by the fluorescence measurement of the expression intensity of BBa_K3078012 was 30% higher than that of wild-type T7 promoters.

Sequence and Features