Difference between revisions of "Part:BBa K5453004"
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                 <p style="color:Gray; padding:0px 30px 10px;">Figure 1:PCR Results Figure. A: Gel electrophoresis image of PCR amplification for sgRNA-pfkA-M, sgRNA-zwf-M. B: Gel electrophoresis image of colony PCR performed on 10 randomly picked colonies from the plate. C: Gel electrophoresis image of plasmid digestion with KpnI and XhoI enzymes.D: Sequencing of the correct plasmids verified by colony PCR and enzyme digestion.</p>
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                 <p style="color:Gray; padding:0px 30px 10px;">     Figure 1:Schematic diagram of the R6k-dcas9-pfkA-zwf-M plasmid..</p>
 
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==Experiments==
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First, we amplified the sgRNA-zwf-M and sgRNA-pfkA-M fragments and purified the amplified products using a gel extraction method. Then, utilizing the Golden Gate assembly technique, which relies on type IIS restriction enzymes, we ligated the sgRNA-zwf-M and sgRNA-pfkA-M fragments into a dCas9-containing vector. After transforming the ligation products into DH5α competent cells, we performed colony PCR, restriction enzyme analysis, and sequencing validation, ultimately successfully constructing the R6K-dCas9-zwf-pfkA-M plasmid.

Revision as of 11:23, 29 September 2024


dcas9-sgRNA-pfkA-lac operator-lacI promoter-sgRNA-zwf-lac operator-lacI promoter

dcas9-sgRNA-pfkA-lac operator-lacI promoter-sgRNA1-zwf-lac operator-lacI promoter

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1096
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3375
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design

We employed the CRISPRi technology to simultaneously inhibit the pfkA and zwf genes, introducing random mutations at the 7th and 8th nucleotide positions of their sgRNA, and constructed an R6K-dCas9-zwf-pfkA mutant library using Golden Gate technology.

Figure 1:Schematic diagram of the R6k-dcas9-pfkA-zwf-M plasmid..

Experiments

First, we amplified the sgRNA-zwf-M and sgRNA-pfkA-M fragments and purified the amplified products using a gel extraction method. Then, utilizing the Golden Gate assembly technique, which relies on type IIS restriction enzymes, we ligated the sgRNA-zwf-M and sgRNA-pfkA-M fragments into a dCas9-containing vector. After transforming the ligation products into DH5α competent cells, we performed colony PCR, restriction enzyme analysis, and sequencing validation, ultimately successfully constructing the R6K-dCas9-zwf-pfkA-M plasmid.