Coding

Part:BBa_K3580200:Design

Designed by: Ryota miyachi   Group: iGEM20_Waseda   (2020-10-23)
Revision as of 07:58, 27 October 2020 by Kanta (Talk | contribs) (→‎References)


Alanine racemase with 6xHis-Tag


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 367
    Illegal NotI site found at 1121
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 291
    Illegal BamHI site found at 21
    Illegal BamHI site found at 993
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 409
    Illegal AgeI site found at 709
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 166


Design Notes

BBa_K3580200 is an AR sequence with 6xHis tag. This part includes the same sequence of AR as BBa_K1172901, but it is new having 6xHis sequence. 6xHis has the same amino acid sequence with BBa_K1223006 at the 5’ end but some codons are different. This 6x His-tag facilitates the purification of alanine racemase by affinity purification. This part’s sequence does not include BioBrick(RFC 10) sequences.  

In our project, we inserted this part between bacteriophage T5 promoter for E. coli RNA polymerase and lambda t0 terminator of pCA24N(-gfp) planmid. The sequence of this promoter is partly consistent with part BBa_K592008, T5-lac Promoter. This allows for the induction of expression by IPTG.


Source

This is a new part including coding region encoded in part BBa_K1172901.

By using His-Tag, we can purify the alanine racemase.

References

[Walish 1989] Walsh, C. T. (1989). Enzymes in the D-alanine branch of bacterial cell wall peptidoglycan assembly. The Journal of Biological Chemistry, 264(5), 2393-2396.

[Azam 2016] Azam, M. A., & Jayaram, U. (2016). Inhibitors of alanine racemase enzyme: A review. Journal of Enzyme Inhibition and Medicinal Chemistry, 31(4), 517-526. doi:10.3109/14756366.2015.1050010