Part:BBa_K3868099

pTarget-Donor

pTarget-donnr plasmid contains pj23119, sgRNA-CM etc.The pCas / pTardet-donnr complex can make the related lacI operator gene in DE3 region replaced by the antibiotic gene of chloramphenicol (CM) . Importantly, the gene of CM can also be used as secondary screening pressure to remove false positive strains.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 182
  Illegal XbaI site found at 188
  Illegal SpeI site found at 73
  Illegal PstI site found at 200
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 182
  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 50
  Illegal SpeI site found at 73
  Illegal PstI site found at 200
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 182
  Illegal BglII site found at 212
  Illegal XhoI site found at 236
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 182
  Illegal XbaI site found at 188
  Illegal SpeI site found at 73
  Illegal PstI site found at 200
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 182
  Illegal XbaI site found at 188
  Illegal SpeI site found at 73
  Illegal PstI site found at 200
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

        In this part, we still takes the dual plasmid expression system for genome editing. The sgRNA on the pTarget+Donor was designed to target the gene of CM (Fig.7A). The pCas and pTarget+Donor plasmids were co-transformed into BL21 (DE3)-CM via electroporation, then the single colonies were cultivated in LB medium with CM and non-CM, respectively. The transformants that cannot grow under CM condition were considered correct ant used for further sequencing (Fig.7B).

Results

         During construction of the library, 90 single colonies were randomly selected for sequencing. It was found that 48 variants with different RBS sequence were identified from 90 samples, with an editing efficiency of only 53%. However, it is noteworthy that the transformants were grown on the solid medium for longer time, the reproducibility of the results gradually increased, and majority of variant RBS sequences became GAAAAAAG (Fig.4), probably due to the continuous base editing in the transformants. The above results show that although CBE possesses the advantages of simplicity and rapidity, editing results and efficiency applied in BL21 (DE3) are not sufficiently stable.

Fig 7. A. The schematic diagram of pTarget+Donor and the composite part of BBa_K3868099. B. The whole process of constructing the library of BL21 (DE3)-derived variant strains using CRISPR/Cas9. C. The partial sequences and the G and A abundance variation maps of RBS variants obtained by CRISPR from three batches respectively.

Reference

Schlegel S, Löfblom J, Lee C, Hjelm A, Klepsch M, Strous M, Drew D, Slotboom DJ, De Gier JW, Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21 (DE3). Journal of molecular biology. 2012; 423: 648–659.