Part:BBa_K4630202:Design

pCasop-stgRNA(1+2+3)

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 679
  Illegal EcoRI site found at 2517
  Illegal EcoRI site found at 6611
  Illegal XbaI site found at 10720
  Illegal SpeI site found at 671
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 679
  Illegal EcoRI site found at 2517
  Illegal EcoRI site found at 6611
  Illegal NheI site found at 2276
  Illegal NheI site found at 9920
  Illegal SpeI site found at 671
  Illegal NotI site found at 696
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 679
  Illegal EcoRI site found at 2517
  Illegal EcoRI site found at 6611
  Illegal BglII site found at 5378
  Illegal BamHI site found at 1043
  Illegal BamHI site found at 4555
  Illegal BamHI site found at 6545
  Illegal BamHI site found at 12241
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 679
  Illegal EcoRI site found at 2517
  Illegal EcoRI site found at 6611
  Illegal XbaI site found at 10720
  Illegal SpeI site found at 671
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 679
  Illegal EcoRI site found at 2517
  Illegal EcoRI site found at 6611
  Illegal XbaI site found at 10720
  Illegal SpeI site found at 671
  Illegal NgoMIV site found at 9173
  Illegal AgeI site found at 878
  Illegal AgeI site found at 6380
  Illegal AgeI site found at 8044
  Illegal AgeI site found at 8920
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 860
  Illegal SapI site found at 6362
  

Golden Gate Assembly and restriction cloning

Though we get a useful pCasop, we intend to make the entire device more compact and better contribute to the synthetic biology. In detail, we design Golden Gate Assembly primers for ligation of the Cas9 coding sequence and the Lambda Red, araC sequence (fig 9a). Considering the multi-cloning site on the pCDF-Duet1 backbone, we attempt to add XhoI and NotI prefixes on the ligated Cas-Lambda sequence (fig 9b). Then the segment can be ligated to the plasmid by restriction cloning. We re-organize them in one integrated plasmid (fig 9c).

Fig. 1 Design of the integrated plasmid, using Golden Gate Assembly and restriction cloning
(a) Using 5’-modified primers, we flank the target sequence with restriction site.
(b) XhoI and NotI recognition sites are present in the multi-cloning site of the recorder plasmid.
(c) The constructed plasmid.

We successfully amplified the target sequence and finished the Golden Gate assembly (fig 10). Unfortunately, due to the limitation of experimental materials, we can only get the two enzymes from different companies, causing the buffer incompatibility. We found it hard to ligate the segment and backbone by restriction cloning.

Fig. 2 The preparation of the target sequence
(a) The white hollow arrow heads indicate target bands. The Cas9 and Lambda fragments are expected to be 4619 and 3434bp, respectively.
(b) The white hollow arrow heads indicate target bands. The primers are designed to cover the sequence of the ligated site. The products are expected to be 2085 bp.

From the plasmid construction, we confirm that the set of Golden Gate Assembly primers are quite versatile and the risk of restriction buffer incompatibility should be noticed.

Three-segment Golden Gate Assembly

This turn, we decide to amplify the two segment and the backbone using Golden Gate Assembly primers (see also experiments, primer list) (fig 11).

Fig. 3 Design of the integrated plasmid
(a) Prefix design for the three-segment Golden Gate Asembly.
(b) The constructed plasmid.

The three segments were successfully amplified, and we performed Golden Gate Assembly (fig 12a-c).

Fig. 4 Integrated plasmid construction
The prepared Cas, Lambda and backbone DNA sample were used as positive control, as the paired samples differ at several 10 bp.
(a) The preparation of backbone. The white hollow arrowhead indicates the target band. The segments are expected to be 4197 bp.
(b) The preparation of Cas and Lambda segments. The white hollow arrowheads indicate the target band. The Cas and Lambda segments are expected to be 4593 and 3399 bp, respectively.
(c) The verification of the plasmid. The primers are designed to cover the ligation site.

Design Notes

This plasmid is suitable for our project CRISPRrporter. If you need a plasmid that expresses the Cas9 protein and Lambda Red recombinases, please refer to BBa_K4630201

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).