We call this updated system as pEcCas/pEcgRNA.  We found that gRNA-pMB1 indeed showed a slightly higher leaky expression in the pCas/pTargetF system compared with pEcCas/pEcgRNA.  We also confirmed that genome editing can successfully be performed in BL21(DE3) by pEcCas/pEcgRNA with high efficiency.  The application of pEcCas/pEcgRNA was then expanded to the E. coli B strain BL21 StarTM (DE3), K-12 strains MG1655, DH5α, CGMCC3705, Nissle1917, W strain ATCC9637, and also another species of Enterobacteriaceae, Tatumella citrea DSM13699, without any specific modifications.  The entire protocol (including plasmid construction, editing, electroporation and mutant verification, and plasmid elimination) took only $\sim$5.5 days per round in the pEcCas/pEcgRNA system, whereas it took $\sim$7.5 days in the pCas/pTargetF system. 
pEcCas/pEcgRNA