Coding

Part:BBa_K4011003:Design

Designed by: Peijia Lai   Group: iGEM21_LINKS_China   (2021-10-19)
Revision as of 03:43, 21 October 2021 by Peggie (Talk | contribs)


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

1. All codons were optimized for E. coli based on E. coli codon bias.

2. We used a rigid linker with the protein sequence EAAAKEAAAK to fuse Fre and SttH.

3. We first added his-tag to Fre-SttH, however only to find that its expression is very low and insoluble in both T7 and ptac systems.

4. We first attempted the T7 system, which is commonly used in E. coli BL21(DE3) to express Fre-SttH. However we found that its expression and solubility to be fairly low, we then switched to the ptac system which can be used in all E. coli strains.

5. The ptac system allows us to express Fre-SttH in a knockout TnaA strain DH5α(TnaA is naturally expressed in E. coli, giving it its characteristic scent) so that our natural trp will not be converted to indole without being halogenized by 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