Coding

Part:BBa_K3017011

Designed by: Luk Hau Ching   Group: iGEM19_Hong_Kong_HKUST   (2019-10-14)
Revision as of 06:40, 17 October 2019 by HannahLuk (Talk | contribs)


dCas9-6XHis

The CRISPR associated protein, in our case, dCas9, is a non-specific deactivated endonuclease. dCas9 is catalytically dead with mutations D10A and H840A. It binds to the target protein, when coupled with single-guide RNA, but does not induce any DNA breakage as in Cas9 enzyme.

In our project, Combined CRISPRi and antisense RNA Toggle Switch, dCas9 plays a central role in CRISPR interference. dCas9 acts as an agent of steric hindrance to block mRNA polymerization to repress expression levels of target genes.

Since we failed multiple times to transform a previous dCas9 part from the iGEM kit plate, we sought help from Professor Ho Yi Mak from our university, who is also working with dCas9. The plasmid Professor Mak offered contains a Streptococcus pyogenes dCas9 gene optimized for mammalian cells but also functional in E.coli. We designed multiple PCR overhang primers to make the dCas9 gene compatible with the standard assembly so that we could clone it into RFC10 plasmids in further steps.

The dCas9 protein is tagged with 6XHis at the C-terminus (BBa_K3017011) for easy characterization. In BBa_K3017029, parts BBa_K608002 and BBa_B0015 are added before and after dCas9-6XHis respectively by PCR overhangs. As the dCas9 protein coding region contains intrinsic PstI cut sites, we could not add an iGEM suffix after the part. Instead, we added iGEM prefix and SpeI and SalI cut sites as a suffix for further digesting-ligation steps.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 790
    Illegal PstI site found at 2212
    Illegal PstI site found at 2416
    Illegal PstI site found at 2446
    Illegal PstI site found at 3658
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 790
    Illegal PstI site found at 2212
    Illegal PstI site found at 2416
    Illegal PstI site found at 2446
    Illegal PstI site found at 3658
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 251
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 790
    Illegal PstI site found at 2212
    Illegal PstI site found at 2416
    Illegal PstI site found at 2446
    Illegal PstI site found at 3658
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 790
    Illegal PstI site found at 2212
    Illegal PstI site found at 2416
    Illegal PstI site found at 2446
    Illegal PstI site found at 3658
    Illegal NgoMIV site found at 1078
    Illegal NgoMIV site found at 2182
    Illegal NgoMIV site found at 2255
    Illegal NgoMIV site found at 2740
    Illegal NgoMIV site found at 3649
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None