Intermediate

Part:BBa_K3127995

Designed by: Zhaolong Li   Group: iGEM19_SDU_CHINA   (2019-10-16)


Advanced RBS for CcaS

Improved Construction intermediate


Usage and Biology

Our team has optimized the BBa_S04615 part. This part consists of the promoter B0034 and the BBa_K592001.

In 2018, the SDU-CHINA team optimized the K592001 part to reduce its leakage under the red light. We have improved its promoters this year, which has further bettered the performance of this component.

The component BBa_K2627000 proved the progress of the optimized CcaS by sufficient characterization, so we directly replaced K592001 in BBa_S04615 with the optimized K2627000 when characterizing this component. In 2014, Schmidl S R , Sheth R U, Wu A, et al. optimized CcaS by constructing an RBS library. This document has fully proven that the performance of components with optimized RBS is better than S04615. In 2019, our team SDU-CHINA has further improved the components based on them. We have introduced site-directed mutagenesis to the improved promoter, further bettering the performance based on all previous studies.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 623
    Illegal NheI site found at 680
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1396
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

Characaterization of BBa_K3127995

The plasmid carrying our RBS is called 5-10#10. There is less fluorescence intensity than #10 under light of 640-650 nm wavelength.
The following figure shows the relative fluorescence intensity induced by the light of 640-650 nm wavelength(green light) and the light of 530-535 nm wavelength(red light).

Figure 1: Characterization of this old part in E.coli BW25113 in M9 medium whose carbon source is glucose.
Figure 2: Characterization of this improved part in E.coli BW25113 in M9 medium whose carbon source is glucose.
Figure 3: Characterization of this old part in E.coli TOP10 in M9 medium whose carbon source is glucose.
Figure 7: Characterization of this improved part in E.coli TOP10 in M9 medium whose carbon source is glucose.

Reference

[1]Schmidl, S. R., Sheth, R. U., Wu, A., & Tabor, J. J. (2014). Refactoring and Optimization of Light-Switchable Escherichia coli Two-Component Systems. ACS Synthetic Biology, 3(11), 820-831.

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