Lethal endotoxin (ccdB) based counterselection improved the efficiency of sequential gene editing in Escherichia coli

Abstract

The chemicals used in this study are analytical grade. The IPTG inducer and L- ( +) arabinose were obtained from Macklin. The restriction enzymes, T5 exonuclease (1000 U), PrimeSTAR Max DNA Polymerase, and 2 × Taq polymerase were provided by Takara. The primers were synthesized by Wuhan Tsingke Biotech Co., Ltd.|||PCR reactions were performed in 50 µL volumes containing 50 ng of template DNA, 300 pmol of each oligonucleotide primer, 25 µL of PrimeSTAR Max DNA Polymerase, 2 × Taq polymerase. DNA sequencing was conducted by Wuhan Tsingke Biotech Co., Ltd. or by DynaScience.|||The resulting colonies were screened through colony PCR with the primer pairs pSim6-ccdB-yz-F/R. The colony PCR verified plasmids were then isolated and sequenced (Wuhan Tsingke Biotech Co., Ltd.). The resulting plasmid with right sequence was designated as pSim6-PL-ccdB.|||After overnight incubation on LB-chlortetracycline plates, the resulting colonies were verified via colony PCR using the primers N20 (cstA)-yz-F/R, N20 (ppsA)-yz-F/R, and N20(csc)-yz-F/R, respectively. Upon successful colony PCR verification, plasmids were isolated and sequenced (Wuhan Tsingke Biotech Co., Ltd.). The plasmid with right sequence was designated as pTargetF-tcr-PL-ccdB-N20 (cstA), pTargetF-tcr-PL-ccdB-N20 (ppsA), and pTargetF-tcr-PL-ccdB-N20 (csc), respectively.|||After removing 4 mL of the supernatants, the pellets were resuspended and diluted 100 folds with SOC broth. 100 µL of the diluted cells were spread onto LB-(kanamycin + chlortetracycline) plates and incubated overnight at 30 °C. The colonies obtained were verified through colony PCR with the cstA-YZ-F/R primers and DNA were sequenced (Wuhan Tsingke Biotech Co., Ltd). The strain with the expected cstA deletion was designated as HBUT-P2-∆cstA.|||The digested DNA mixture was subsequently transformed into E. coli DH5α (Chang et al. 2017). The resulting kanamycin resistance colonies were verified by colony PCR with the donor-yz-F/R primers. The PCR verified plasmid were then sequenced (Wuhan Tsingke Biotech Co., Ltd). The plasmid with the correct sequence was designated pET-donor.