Composite

Part:BBa_K5159029

Designed by: Tianyue WU   Group: iGEM24_HKUST   (2024-10-02)

KU70 Knockout Cassette

This part was designed with the 5’ and 3’ homologous arms of Yarrowia lipolytica KU70 gene flanking a LEU2 selection marker expression cassette, which can be released by the Cre-loxP system after use. The construct knocks out part of the native KU70 gene, replacing it by the LEU2 expression cassette through homologous recombination (HR). KU70 encodes a protein in charge of the double-strand break (DSB) repair in Yarrowia lipolytica, which enables non-homologous end-joining (NHEJ), a genomic integration mechanism happening in Yarrowia lipolytica at a much higher frequency than HR. Knocking out KU70 can increase the chance for subsequent integration via HR by several folds. As further knockouts via HR are involved, for example, knocking out PEX10 in our project, constructing a KU70 knockout strains would significantly increase the success rate of engineering.

As before the knockout Yarrowia lipolytica still shows a higher tendency of utilizing NHEJ instead of HR, two 1000 bp homologous arms in length are necessary for the efficient knockout to occur.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1035
    Illegal PstI site found at 3675
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1035
    Illegal PstI site found at 3675
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1035
    Illegal BglII site found at 3267
    Illegal XhoI site found at 1981
    Illegal XhoI site found at 2010
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1035
    Illegal PstI site found at 3675
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1035
    Illegal PstI site found at 3675
    Illegal NgoMIV site found at 40
    Illegal NgoMIV site found at 1123
    Illegal AgeI site found at 2595
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1641
    Illegal BsaI site found at 1924
    Illegal BsaI.rc site found at 1839
    Illegal SapI site found at 140
    Illegal SapI site found at 2392
    Illegal SapI.rc site found at 718

Reference

[1] Jang I-S, Yu B. J, Jang J. Y, Jegal J, Lee J. Y. (2018). Improving the efficiency of homologous recombination by chemical and biological approaches in Yarrowia lipolytica. PLoS ONE 13(3): e0194954. https://doi.org/10.1371/journal.pone.0194954


[edit]
Categories
Parameters
None