Part:BBa_K3385073

E_coli_backbone

About the part: The part was obtained as part of the CRISPR cloning vector pFC330 [1] and pFC902 [2]. The E. coli backbone used in pFC330 and pFC902 contains ori and ampR for selection on growth medium containing ampicillin.

Plasmid map of pFC330[1].


Plasmid map of pFC902[2].

Functionality: This E.coli backbone can be used to select and propagate the CRISPR vectors FC330 [1] and pFC902 [2]. The selection mechanism involves ampicilin resistance, while propagation is ensured with the origin of replication (ori).

Results: Below is a picture showing colony PCR's of transformed E.coli. It shows integration and propagation in E.coli with succesfull bands at 1125 bp. Positve control used were the pCF902[2] vector yielding a band at 989 and a negative control with MQ water yielding no band.

Colony PCR's of transformed E.coli strains harbouring CRISPR plasmids. Succesfull integration with USER assembly is observed as a band at 1125 bp. Positive control include the pCF902[2] without USER assembly which is observed as a band at 989 bp, while the negative control included were MQ water and shows no band.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal XbaI site found at 63
  Illegal XbaI site found at 2807
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal NotI site found at 20
  Illegal NotI site found at 2846
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal XbaI site found at 63
  Illegal XbaI site found at 2807
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal XbaI site found at 63
  Illegal XbaI site found at 2807
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  

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