Part:BBa_K4630201:Design

The optimized pCas

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1344
  Illegal EcoRI site found at 7542
  Illegal EcoRI site found at 11785
  Illegal SpeI site found at 5392
  Illegal PstI site found at 11075
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1344
  Illegal EcoRI site found at 7542
  Illegal EcoRI site found at 11785
  Illegal NheI site found at 1103
  Illegal SpeI site found at 5392
  Illegal PstI site found at 11075
  Illegal NotI site found at 5711
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1344
  Illegal EcoRI site found at 7542
  Illegal EcoRI site found at 11785
  Illegal BglII site found at 4205
  Illegal BamHI site found at 3382
  Illegal BamHI site found at 7476
  Illegal BamHI site found at 11664
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1344
  Illegal EcoRI site found at 7542
  Illegal EcoRI site found at 11785
  Illegal SpeI site found at 5392
  Illegal PstI site found at 11075
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1344
  Illegal EcoRI site found at 7542
  Illegal EcoRI site found at 11785
  Illegal SpeI site found at 5392
  Illegal PstI site found at 11075
  Illegal NgoMIV site found at 10624
  Illegal AgeI site found at 7311
  Illegal AgeI site found at 8973
  Illegal AgeI site found at 11499
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4231
  Illegal BsaI.rc site found at 4219
  Illegal SapI site found at 7293
  Illegal SapI site found at 10564
  Illegal SapI site found at 10774
  Illegal SapI site found at 11481
  

As the length of the segments vary, we decided to remove redundant part step by step, especially the functional sgRNA. We designed primers for the backbone to delete the sgRNA sequence using Gibson Assembly and Golden Gate Assembly (fig 1).

Fig. 1The sgRNA-removal design of pCas optimization

We successfully amplified the target sequence of pCas (fig 2a). Subse-quently, we employed Golden Gate Assembly. Then sequencing result unveiled that the sgRNA part of pCas had been removed (fig 2c).

Fig. 2The result of sgRNA deletion in pCas
(a) The white hollow arrow heads indicate target bands. The long fragments for Gibson Assembly and Golden Gate Assembly are expected to be 11610 and 11744 bp, respectively. The regular PCR procedure is effective.
(b) The white hollow arrow heads indicate target pCasop bands. The primers were designed to amplify sequence covering the omitted sgRNA.
(c) Sequencing results were aligned with the original pCas sequence. There is a gap at sgRNA.
(d) Sequencing results were aligned with the designed pCasop sequence.

In the next step, we intend to remove the homologous arm of poxb gene (Fig3). We designed to clone the extremely long fragment using a pair of unique primers and link it into a closed loop using Golden Gate Assembly. Regrettably, only the ultra-long fragment was successfully cloned (Fig4) within the given time constraints, while the connection failed.

Fig. 3The design of Removing Homologous Arm Sequence

Fig. 4 The result of PCR fragment, cloned by removing HA primers

Lanes 1 to 4 consist of parallel groups of amplified ultra-long fragments, with a fragment length of 11744 bp.

Design Notes

The plasmid is temperature sensitive.

As the length of the segments vary, we decided to remove redundant part step by step, especially the functional sgRNA. We designed primers for the backbone to delete the sgRNA sequence using Gibson Assembly and Golden Gate Assembly (fig 1).

Source

Zhao, D., Yuan, S., Xiong, B. et al. Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Fact 15, 205 (2016).

References

Zhao, D., Yuan, S., Xiong, B. et al. Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Fact 15, 205 (2016).