Plasmid

Part:BBa_K3385000

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


pFC330

About the part: This is the Cas9 vector, pFC330, which contains the A. niger codon optimised Cas9 for gRNA mediated knockout, the selection marker pyrG, and the plasmid replicator AMA1 [1]. pFC330 was constructed by Uffe Mortensen.

The plasmid also contains the PacI/Nt.BbvCI cassette, which enables restriction digestion of the plasmid followed by integration of sgRNA's flanked by nuclear-localized promoters and terminators to ensure double-stranded breaks.


Plasmid map of pFC330[1].


Functionality: The sgRNA efficiency has been accessed through the technique to assess protospacer efficiency (TAPE) [2]. 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 7744
    Illegal EcoRI site found at 8007
    Illegal EcoRI site found at 9652
    Illegal EcoRI site found at 11273
    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 11307
    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 12111
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1720
    Illegal EcoRI site found at 3997
    Illegal EcoRI site found at 7744
    Illegal EcoRI site found at 8007
    Illegal EcoRI site found at 9652
    Illegal EcoRI site found at 11273
    Illegal NheI site found at 12767
    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 12111
    Illegal NotI site found at 12758
    Illegal NotI site found at 12836
    Illegal NotI site found at 15662
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1720
    Illegal EcoRI site found at 3997
    Illegal EcoRI site found at 7744
    Illegal EcoRI site found at 8007
    Illegal EcoRI site found at 9652
    Illegal EcoRI site found at 11273
    Illegal BglII site found at 10902
    Illegal BamHI site found at 8818
    Illegal BamHI site found at 11162
    Illegal BamHI site found at 11313
    Illegal BamHI site found at 11849
    Illegal XhoI site found at 2261
    Illegal XhoI site found at 2978
    Illegal XhoI site found at 3456
    Illegal XhoI site found at 10177
    Illegal XhoI site found at 11709
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1720
    Illegal EcoRI site found at 3997
    Illegal EcoRI site found at 7744
    Illegal EcoRI site found at 8007
    Illegal EcoRI site found at 9652
    Illegal EcoRI site found at 11273
    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 11307
    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 12111
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1720
    Illegal EcoRI site found at 3997
    Illegal EcoRI site found at 7744
    Illegal EcoRI site found at 8007
    Illegal EcoRI site found at 9652
    Illegal EcoRI site found at 11273
    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 11307
    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 12111
    Illegal NgoMIV site found at 2998
    Illegal NgoMIV site found at 6839
    Illegal AgeI site found at 9125
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5591
    Illegal BsaI site found at 11902
    Illegal BsaI.rc site found at 126
    Illegal BsaI.rc site found at 6802
    Illegal BsaI.rc site found at 9823
    Illegal SapI site found at 6786
    Illegal SapI site found at 7182


References:
[1] A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. Nodvig CS, Nielsen JB, Kogle ME, Mortensen UH. PLoS One. 2015 Jul 15;10(7):e0133085. doi: 10.1371/journal.pone.0133085. eCollection 2015. PONE-D-15-11561 [pii] PubMed 26177455

[2] 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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