Composite

Part:BBa_K4887021

Designed by: Duoqing LIN   Group: iGEM23_Shanghai-BioX   (2023-09-19)


Backbone of IbGBSSI knockout system

This part is the backbone of tht knockout system of gene iBGBSSI (BBa_K4887001).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 2106
    Illegal PstI site found at 3528
    Illegal PstI site found at 3732
    Illegal PstI site found at 3762
    Illegal PstI site found at 4974
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 2106
    Illegal PstI site found at 3528
    Illegal PstI site found at 3732
    Illegal PstI site found at 3762
    Illegal PstI site found at 4974
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1567
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 2106
    Illegal PstI site found at 3528
    Illegal PstI site found at 3732
    Illegal PstI site found at 3762
    Illegal PstI site found at 4974
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 2106
    Illegal PstI site found at 3528
    Illegal PstI site found at 3732
    Illegal PstI site found at 3762
    Illegal PstI site found at 4974
    Illegal NgoMIV site found at 1157
    Illegal NgoMIV site found at 1176
    Illegal NgoMIV site found at 2394
    Illegal NgoMIV site found at 3498
    Illegal NgoMIV site found at 3571
    Illegal NgoMIV site found at 4056
    Illegal NgoMIV site found at 4965
  • 1000
    COMPATIBLE WITH RFC[1000]

12.5 Results:

(1) Construction of the backbone vector of sgRNA (IbGBSSI)

The backbone plasmid psgR-Cas9-At were digested and the products were ligated with sgRNA oligos using T4 ligase, and obtained the backbone vector of sgRNA (IbGBSSI): psgR-Cas9- sgRNA(IbGBSSI). To confirm the correctness of the vector, 1/2 of the DNA was then transform into bacteria and incubate in LB for 45 min, and was plated in LB containing Ampicillin (Fig. 1). The obtained clones were validated by performing PCR detection for the sgRNA sequence with primers M13F/oligo2 (The sequence of M11F was: 5’-TGTAAAACGA CGGCCAGT-3’). The gel electrophoresis results (Fig. 2) showed that the gene band were approximately 100bp, as expected, indicating that backbone vector of sgRNA (IbGBSSI) was been constructed successfully.
Fig. 1 E. Coli transformed with backbone vector of sgRNA (IbGBSSI)
Fig.2 PCR product of sgRNA (IbGBSSI) in E. Coli transformed with backbone vector of sgRNA (IbGBSSI)


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