Fig. 5
From: Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli
The design and stability of RPGPs pDG-R-X in E. coli. a The modular construction strategy of pDG-R-X series. pKT plasmid was designed for PCR amplification of DNA part 1 and part 2 series. DNA part 1 which contained pDG-R-X backbone and two reversed repeated gRNA scaffolds was amplified by using only one prime. DNA part 2 contained two different promoters followed by a 20-bp spacer sequence, respectively. For the PCR reaction, the 20-bp spacers specific for two targeted loci and another 20-bp overlap sequences for assembly were embedded in primers as a part of insert. Gibson Assembly method was used to assemble these parts into pDG-R1-X or pDG-R2-X series. b Representative PCR results of pDG-R1-100K after re-transformation. c The double restriction enzyme digestion analyses of pDG-R1-100K