Team:DTU-Denmark/Parts

Here is an overview of all of the 76 parts we have used for this years project.

Click on the navigation bar below to see the different parts!

Parts from Professor Uffe Mortensen's Lab

The plasmids pFC330 (Nødvig et. al, 2015) and pFC902 (Nødvig et al., 2018) have built the foundation for our project. pFC330 is a plasmid containing an optimized Cas9 for our project. By coupling pFC330 to gRNA's made using the pFC902 as a template, one is able to make CRISPR-Cas9 mediated double stranded breaks to knock genes out of the genome. These parts have been designed, built and supplied by Uffe Mortensen's Lab. These parts are new to the iGEM registry.

CRISPR plasmid components

Part name Part number Short description
Aspergillus nidulans glycin tRNA BBa_K3385002 tRNA spacer used to cleave transcribed sgRNA.
gRNA backbone BBa_K3385003 gRNA backbone used for Cas9 mediated double-stranded breaks.
Aspergillus fumigatus U3 promoter BBa_K3385004 Aspergillus fumigatus U3 promoter, chosen for it's ability to stimulate sgRNA production.
Aspergillus fumigatus U3 terminator BBa_K3385005 Aspergillus fumigatus U3 terminator chosen for efficiency in generating sgRNA.
Ptef BBa_K3385006 Promoter domesticated from Aspergillus nidulans.
Aspergillus niger Cas9 BBa_K3385007 Cas9 adapted from Streptococcus pyogenes and codon optimized for A. niger . This part is the very base for knocking out genes from the genome and was used for all our knockouts.
pyrG BBa_K3385072 This is the selection marker pyrG, which is detrimental for pyrimidine biosynthesis.
E_coli_backbone BBa_K3385073 This is the E. coli backbone used in pFC330 and pFC902. Includes ori and ampR
NLS BBa_K3385074 It's a nuclear localization signal for Cas9, which ensures that the Cas9 is guided to the nucleus.
PacI/Nt.BbvCI cassette BBa_K3385075 This is the PacI/Nt.BbvCI cassette which enables RE digestion of the pFC330 plasmid to insert the CRISPR constructs.

CRISPR-Cas9 backbone and CRISPR template plasmid

Part name Part number Short description
pFC330 BBa_K3385000 This Cas9 vector contains an A. niger codon optimized Cas9 for gRNA mediated knockout, the selection marker pyrG, and the plasmid replicator AMA1.
pFC902 BBa_K3385001 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.



References

  1. Nødvig, C., Nielsen, J., Kogle, M., & Mortensen, U. (2015). A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PLOS ONE, 10(7). doi:10.1371/journal. Pone.0133085
  2. Nødvig, C., Hoof, J., Kogle, M., Jarczynska, Z., Lehmbeck, J., Klitgaard, D., & Mortensen, U. (2018). Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli. Fungal Genetics And Biology, 115, 78-79. doi: 10.1016/j.fgb.2018.01.004
  3. Cerqueira, G., Arnaud, M., Inglis, D., Skrzypek, M., Binkley, G., & Simison, M. et al. (2014). The Aspergillus Genome Database: multispecies curation and incorporation of RNA-Seq data to improve structural gene annotations. Nucleic Acids Res., 42(1). doi: 10.1093/nar/gkt1029