Part:BBa_K3868095

pCas

pCas plasmid of the Crispr-Cas9 system contains Pcas, Spcas9, paraB, λ-red, paraC and arac etc, which plays a role in cutting DNA double strands.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1572
  Illegal EcoRI site found at 2469
  Illegal EcoRI site found at 3978
  Illegal PstI site found at 3473
  Illegal PstI site found at 3720
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1572
  Illegal EcoRI site found at 2469
  Illegal EcoRI site found at 3978
  Illegal NheI site found at 1331
  Illegal PstI site found at 3473
  Illegal PstI site found at 3720
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1572
  Illegal EcoRI site found at 2469
  Illegal EcoRI site found at 3978
  Illegal BamHI site found at 2408
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1572
  Illegal EcoRI site found at 2469
  Illegal EcoRI site found at 3978
  Illegal PstI site found at 3473
  Illegal PstI site found at 3720
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1572
  Illegal EcoRI site found at 2469
  Illegal EcoRI site found at 3978
  Illegal PstI site found at 3473
  Illegal PstI site found at 3720
  Illegal AgeI site found at 3900
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

        To achieve the editing of the RBS sequence using CBE, a tailored RBS sequence of 5'-GGGGGGGG-3' was integrated into the corresponding site of the T7 RNAP, yielding a starting strain of BL21 (DE3)-RBS8G. Here, the CRISPR/Cas9 system was used to construct the BL21 (DE3)-RBS8G strain. The pCas and pTarget plamids was provided by our PI, Xiao-Man Sun. In the genome of BL21 (DE3), the CCGGATTTACTAACTGGAAG was chosen as N20 sequence, and then the pTarget-8G plasmid was successfully constructed based on the pTarget (Fig. 1).

Fig 1. The Schematic diagram of pCas, pTarget, and the composite part of BBa_K3868095 and BBa_K3868096

Results

         By sequencing, the RBS sequence of T7RNAP on the genome was successfully replaced by GGGGGGGG (Fig. 2), which indicated that the starting strain BL21 (DE3)-RBS8G was successfully constructed.

Fig 2. The sequencing results of the RBS sequence of T7RNAP on the genome in the BL21 (DE3) and BL21 (DE3)-RBS8G strain.

Reference

1. Wang Y, Cheng H, Liu Y, Wen X, Zhang K, Ma Y. In-situ generation of large numbers of genetic combinations for metabolic reprogramming via CRISPR-guided base editing. Nature communications. 2021; 12(1): 1-12.