Plasmid

Part:BBa_K3385001

Designed by: Daniel Bavnhøj   Group: iGEM20_DTU-Denmark   (2020-10-12)


pFC902

About the part: This vector was used as a template for the construction of the CRISPR parts for CRISPR-Cas9 mediated K/O of genes from the A. niger genome, pFC902 [1]. pFC902 was constructed by Uffe Mortensen.


Plasmid map of pFC902[1].


Functionality: This part was used to make biobricks to assemble different pFC330 vectors by making biobricks. Biobricks were purified and assembled with USER assembly.

Biobricks made from the pFC902 vector for later assembly of CRISPR pFC330 vector.


The assembled vector was used to make CRISPR K/O strains with different sgRNA.The sgRNA efficiency has been accessed through the technique to assess protospacer efficiency (TAPE) [1]. A repair oligo is used to mediate homologous recombination, where a highly efficient sgRNA will show no colonies without the repair oligo, while less efficient sgRNA will show a reduced number of colonies.

Results: Below is a picture showing the transformed A. niger. It shows efficient gene deletion when it's transformed with a repair oligo, while the lack of the repair oligo renders the fungus unable to repair the double-stranded break and leads to death.
TAPE showing sgRNA efficiency. As arfA is an essential gene, it did not grow.

To see if the K/O’s were successful, other than looking at macromorphology, tissue PCRs were performed. By the amplification of specific primers, upstream and downstream of the gene, it can be verified if the gene has successfully been knocked out. If it has been knocked out the primers are gonna be closer to each other resulting in a smaller band in the Tissue PCR. However if the gene is still present in the genome, the band size will be the same as the target gene as seen in the table below.

Expected length of each K/O
Targeted gene Expected gene length after K/O Control lenght
ΔchsC 704 bp 1867 bp
ΔaplD 590 bp 3807 bp
ΔracA 709 bp 1920 bp
ΔspaA 672 bp 3528 bp
Δgul-1 545 bp 5022 bp
ΔpkaR 370 bp 1661 bp


Gel picture showing if our K/O's were successful. Tissue PCRs of ΔchsC, ΔaplD, and ΔracA are shown in this picture.


Gel picture showing if our K/O's were successful. Tissue PCRs of ΔspaA, Δgul-1, and ΔpkaR are shown in this picture.


Gel picture showing if our K/O's were successful. Tissue PCRs of ΔchsC and ΔpkaR are shown in this picture.

The results from the Tissue PCR showed that we successfully transformed the plasmid into A. niger.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1720
    Illegal EcoRI site found at 3997
    Illegal EcoRI site found at 6210
    Illegal EcoRI site found at 6361
    Illegal EcoRI site found at 8634
    Illegal EcoRI site found at 8897
    Illegal EcoRI site found at 10542
    Illegal XbaI site found at 665
    Illegal XbaI site found at 1147
    Illegal XbaI site found at 1915
    Illegal XbaI site found at 3802
    Illegal XbaI site found at 4570
    Illegal XbaI site found at 5052
    Illegal XbaI site found at 12197
    Illegal SpeI site found at 82
    Illegal SpeI site found at 93
    Illegal SpeI site found at 457
    Illegal SpeI site found at 589
    Illegal SpeI site found at 713
    Illegal SpeI site found at 820
    Illegal SpeI site found at 831
    Illegal PstI site found at 2864
    Illegal PstI site found at 13001
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1720
    Illegal EcoRI site found at 3997
    Illegal EcoRI site found at 6210
    Illegal EcoRI site found at 6361
    Illegal EcoRI site found at 8634
    Illegal EcoRI site found at 8897
    Illegal EcoRI site found at 10542
    Illegal NheI site found at 6054
    Illegal NheI site found at 13657
    Illegal SpeI site found at 82
    Illegal SpeI site found at 93
    Illegal SpeI site found at 457
    Illegal SpeI site found at 589
    Illegal SpeI site found at 713
    Illegal SpeI site found at 820
    Illegal SpeI site found at 831
    Illegal PstI site found at 2864
    Illegal PstI site found at 13001
    Illegal NotI site found at 13648
    Illegal NotI site found at 13726
    Illegal NotI site found at 16552
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1720
    Illegal EcoRI site found at 3997
    Illegal EcoRI site found at 6210
    Illegal EcoRI site found at 6361
    Illegal EcoRI site found at 8634
    Illegal EcoRI site found at 8897
    Illegal EcoRI site found at 10542
    Illegal BglII site found at 11792
    Illegal BamHI site found at 9708
    Illegal BamHI site found at 12052
    Illegal BamHI site found at 12203
    Illegal BamHI site found at 12739
    Illegal XhoI site found at 2261
    Illegal XhoI site found at 2978
    Illegal XhoI site found at 3456
    Illegal XhoI site found at 11067
    Illegal XhoI site found at 12599
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1720
    Illegal EcoRI site found at 3997
    Illegal EcoRI site found at 6210
    Illegal EcoRI site found at 6361
    Illegal EcoRI site found at 8634
    Illegal EcoRI site found at 8897
    Illegal EcoRI site found at 10542
    Illegal XbaI site found at 665
    Illegal XbaI site found at 1147
    Illegal XbaI site found at 1915
    Illegal XbaI site found at 3802
    Illegal XbaI site found at 4570
    Illegal XbaI site found at 5052
    Illegal XbaI site found at 12197
    Illegal SpeI site found at 82
    Illegal SpeI site found at 93
    Illegal SpeI site found at 457
    Illegal SpeI site found at 589
    Illegal SpeI site found at 713
    Illegal SpeI site found at 820
    Illegal SpeI site found at 831
    Illegal PstI site found at 2864
    Illegal PstI site found at 13001
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1720
    Illegal EcoRI site found at 3997
    Illegal EcoRI site found at 6210
    Illegal EcoRI site found at 6361
    Illegal EcoRI site found at 8634
    Illegal EcoRI site found at 8897
    Illegal EcoRI site found at 10542
    Illegal XbaI site found at 665
    Illegal XbaI site found at 1147
    Illegal XbaI site found at 1915
    Illegal XbaI site found at 3802
    Illegal XbaI site found at 4570
    Illegal XbaI site found at 5052
    Illegal XbaI site found at 12197
    Illegal SpeI site found at 82
    Illegal SpeI site found at 93
    Illegal SpeI site found at 457
    Illegal SpeI site found at 589
    Illegal SpeI site found at 713
    Illegal SpeI site found at 820
    Illegal SpeI site found at 831
    Illegal PstI site found at 2864
    Illegal PstI site found at 13001
    Illegal NgoMIV site found at 2998
    Illegal NgoMIV site found at 7729
    Illegal AgeI site found at 5925
    Illegal AgeI site found at 10015
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5591
    Illegal BsaI site found at 12792
    Illegal BsaI.rc site found at 126
    Illegal BsaI.rc site found at 5883
    Illegal BsaI.rc site found at 7692
    Illegal BsaI.rc site found at 10713
    Illegal SapI site found at 7676
    Illegal SapI site found at 8072


References:
[1] Efficient Oligo nucleotide mediated CRISPR-Cas9 Gene Editing in Aspergilli. Nodvig CS, Hoof JB, Kogle ME, Jarczynska ZD, Lehmbeck J, Klitgaard DK, Mortensen UH. Fungal Genet Biol. 2018 Jan 8. pii: S1087-1845(18)30004-5. doi: 10.1016/j.fgb.2018.01.004. 10.1016/j.fgb.2018.01.004 PubMed 29325827

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