CRISPR-Based Gene Knockout and Protein Display in E. coli

The ΔycgF strain was obtained from the E. coli W1688 strain by knocking out the target gene ycgF using the CRISPR/Cas9 gene-editing technology. The primers used are shown in Table S1 in the supplemental material. The specific operation is as follows. The donor DNA fragment and pTarget plasmid containing the N₂₀ sequence were constructed and introduced into competent E. coli W1688 cells containing the pCas plasmid. Using Kan (50 mg/L) and Str (40 mg/L) as resistance markers, the transformed cells were screened to select the cells lacking ycgF.
To express nMagHigh and pMagHigh on the surface of E. coli, we fused these proteins to the N terminus of OmpX (45 (link)). To insert the nMagHigh gene into the plasmid pET28a, the gene cassettes nMagHigh-linker-OmpX and pMagHigh-linker-OmpX were first synthesized by Tsingke Biotechnology Co., Ltd. Then, using the ClonExpress II one-step cloning kit C112-01 (Vazyme Biotech Co., Ltd.), these genes were inserted into the BglII restriction sites of pET28a-EGFP and pET28a-mCherry, respectively. Thus, plasmids pET28a-EGFP-nMagHigh-OmpX and pET28a-mCherry-pMagHigh-OmpX were obtained. The constructed plasmid was transformed into ΔycgF using the heat shock transformation method, and the transformants were selected using Kan (50 mg/L).