Part:BBa_K5453004
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1096
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3375
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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