Part:BBa_K1585350

glgX targeting plasmid

This plasmid is targeting glgX in the E. coli genome by expressing a single guide RNA (sgRNA) for CRISPR/Cas9 genome editing. It contains a repair template that disrupts the beginning of the glgX coding sequence and introduces a sequence with multiple stop codons.

Usage and Biology

The construct is part of a two-plasmid knockout system published by [http://aem.asm.org/content/81/7/2506.long| Jiang et al]. This part, the pTargetT, contains the sgRNA to target the glgX coding sequence. Additionally, the construct consists of a repair template with two 400 bp homology arms for homology-directed repair. To succesfully knock out the glgX, you need a second plasmid, pCas. It contains the pCas gene as well as a temperature-sensitive repA101ts ori. To increase recombination efficiency in E. coli, it also expresses lambda-Red genes behind an inducible arabinose-promoter. Upon induction with IPTG, a sgRNA is transcribed to cut the pMB1 ori of the pTargetT.

After cloning both the targeting plasmid and the pCas plasmid in BL21 Gold (DE3) and inducing with arabinose, the Cas9 gene is expressed. Guided by the sgRNA, it cleaves the double strand at the beginning of the glgX gene. The succesful genome editing could be verified by sequencing.

Genomic region and sequencing chromatograms of BL21 Gold (DE3) ΔglgX The sequencing results show that the "IGEM AACHEN MULTISTOP" sequence disrupts the gene. This insert contains a stop codon in each possible frame. The picture demonstrates the functionality of BBa_K1585350.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 167
  Illegal XbaI site found at 173
  Illegal SpeI site found at 58
  Illegal PstI site found at 185
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 167
  Illegal NheI site found at 35
  Illegal SpeI site found at 58
  Illegal PstI site found at 185
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 167
  Illegal BglII site found at 1015
  Illegal BamHI site found at 23
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 167
  Illegal XbaI site found at 173
  Illegal SpeI site found at 58
  Illegal PstI site found at 185
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 167
  Illegal XbaI site found at 173
  Illegal SpeI site found at 58
  Illegal PstI site found at 185
  
• 1000
  COMPATIBLE WITH RFC[1000]