Coding

Part:BBa_K4011003:Design

Designed by: Peijia Lai   Group: iGEM21_LINKS_China   (2021-10-19)
Revision as of 14:10, 19 October 2021 by Registry (Talk | contribs)

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Fre-SttH


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 576
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 948
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 424


Design Notes

We use a rigid linker with the protein sequence EAAAKEAAAK to fuse fre and sttH. SttH is a trp-6-haloganese that requires FADH2 as a cofactor to convert trp into 6-X-trp, and is highly insoluble in E. coli. Therefore, Fre, a highly-soluble flavin reductase which reduces FAD to FADH2 from E. coli, is fused with SttH as a N-terminal soluble tag, enabling the protein to become soluble and eliminating the need for costly FADH2 cofactors to be added.

To express Fre-SttH, we first attempted the T7 system, commonly used in E. coli BL21(DE3). We expressed histag-Fre-SttH using the T7 system with a lacO promoter, but found both its expression and solubility to be fairly low, thus making it unsuitable for our project.

Because we needed a knockout TnaA (TnaA is naturally expressed in E. coli, giving it its characteristic scent) strain to express Fre-SttH so that our natural trp will not be converted to indole without being halogenized by Fre-SttH, we switched from the T7 system to the ptac system. The ptac system, unlike the T7 system which can only be used in E. coli BL21, can be used in all E. coli strains. Our knockout strain was supplied in E. coli DH5α, courtesy of Sha Zhou, in which we constructed ptac-histag-Fre-SttH and ptac-Fre-SttH.




Source

Fre-SttH is composed of two separate domains, fre and sttH. Fre is from E.coli and SttH is from Streptomyces toxytricini.

References