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Revision as of 09:01, 19 October 2021


pTarget-8G

pTarget-8G plasmids contains pj23119 and sgRNA-8G etc, which can target the RBS sequence of T7RNAP for Cas9 expression.

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

        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.