2.1 Construction of strains and plasmids
All plasmids used in this study are listed in Supporting information
Table S1, and the primers are listed in Table S2. E. coli strain
DH5α was used as the cloning host. The GDH plasmids variants with
site-directed mutations and deletions of the RBS site (AAGGAG) were
constructed using megaprimer PCR. The modified pET plasmids with RK2
replicon were constructed by standard restriction cloning based on
Gibson assembly. All mutant strains were generated from BL21 (DE3) using
CRISPR-Cas9 technology (Jiang et al., 2015). The template DNA with
1,000bp homologous arms was prepared by PCR. Strains harboring the pCas
plasmid were transformed with the pTarget plasmid and the template DNA
by electroporation (1.85 kV, 200 Ohm, 25 uF) and regenerated for 1 h in
1 mL LB medium at 37 ℃. The mutants were confirmed by colony PCR.
Correct colonies were then screened and the plasmids cured.