Protein_Domain

Part:BBa_K4618502:Design

Designed by: PO-YANG PENG   Group: iGEM23_CSMU-Taiwan   (2023-10-11)


bpuI + NG catcher


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 825
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 825
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 825
    Illegal XhoI site found at 7
    Illegal XhoI site found at 164
    Illegal XhoI site found at 794
    Illegal XhoI site found at 1034
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 825
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 825
    Illegal AgeI site found at 816
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1132
    Illegal BsaI.rc site found at 1230
    Illegal BsaI.rc site found at 1656


Design Notes

This sequence is combined with bpul and NGCacther. The function of bpul is degradation of textile dyes. And the function of NGCatcher is used to bind the tag which combine with other protein. This sequence is derived from Functional expression enhancement of Bacillus pumilus CotA-laccase mutant WLF through site-directed mutagenesis and Tailoring the Tag/Catcher System by Integrating Covalent Bonds and Noncovalent Interactions for Highly Efficient Protein Self-Assembly. However, we did not directly employ the sequences documented in the literature. We revise the sequence to let it fit E.coli more. Besides, incorporating the existing bpul to remove the stop codon, eliminating the catcher's methionine, allowing the concatenation of both sequences into a single protein. Then we entrusted Twist Bioscience with its synthesis and subsequent cloning into a plasmid. We use this part in the biosafety level 1 laboratory. Because we used E.coli system to express this part, so we can’t promise it can be expressed in other system. Lastly, we employ SDS-PAGE to verify the accuracy of expressed protein's molecular weight.



Source

  GenBank: KF040050.1


References

Paper:Functional expression enhancement of Bacillus pumilus CotA-laccase mutant WLF through site-directed mutagenesis Author links open overlay panel Quan Luo, Yu Chen, Jing Xia, Kai-Qiang Wang, Yu-Jie Cai, Xiang-Ru Liao, Zheng-Bing GuanReceived 27 April 2017, Revised 12 July 2017, Accepted 29 July 2017, Available online 14 September 2017, Version of Record 7 December 2017.