Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (12): 252-264.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0618
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WANG Kai-kai(), WANG Xiao-lu, SU Xiao-yun(), ZHANG Jie()
Received:
2021-05-11
Online:
2021-12-26
Published:
2022-01-19
Contact:
SU Xiao-yun,ZHANG Jie
E-mail:kkw_2012@163.com;suxiaoyun@caas.cn;zhangjie09@caas.cn
WANG Kai-kai, WANG Xiao-lu, SU Xiao-yun, ZHANG Jie. Optimization and Application of Double-plasmid CRISPR-Cas9 System in Escherichia coli[J]. Biotechnology Bulletin, 2021, 37(12): 252-264.
菌株Strain/质粒 Plasmid | 功能 Function | 来源 Source |
---|---|---|
菌株Strain | ||
大肠杆菌Escherichia coli Top10 | 克隆宿主 | 天根 |
MG1655(DE3) | 基本菌株 | 本研究 |
MG1655-∆pfkA | 来源于MG1655(DE3),pfkA缺失突变株 | 本研究 |
MG1655-∆pfkB | 来源于MG1655(DE3),pfkB缺失突变株 | 本研究 |
MG1655-∆zwf | 来源于MG1655(DE3),zwf缺失突变株 | 本研究 |
MG1655-∆ABE∷glpK | 来源于MG1655(DE3),nagABE基因位点替换为glpK的突变株 | 本研究 |
质粒 Plasmid | ||
pRed_cas9_recA_poxb300 | Cas9供体质粒 | MolecularCloud |
p∆pfkA | pfkA同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统 | 本研究 |
pRA∆pfkA | pfkA敲除质粒,用于单质粒CRISPR-Cas9系统 | 本研究 |
p∆pfkB | pfkB同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统 | 本研究 |
pRA∆pfkB | pfkB敲除质粒,用于单质粒CRISPR-Cas9系统 | 本研究 |
p∆zwf | zwf同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统 | 本研究 |
pRA∆zwf | zwf敲除质粒,用于单质粒CRISPR-Cas9系统 | 本研究 |
p∆ABE∷glpK | nagABE同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统 | 本研究 |
pRA∆ABE∷glpK | nagABE敲除质粒,用于单质粒CRISPR-Cas9系统 | 本研究 |
Table1 Strains and plasmids used in this study
菌株Strain/质粒 Plasmid | 功能 Function | 来源 Source |
---|---|---|
菌株Strain | ||
大肠杆菌Escherichia coli Top10 | 克隆宿主 | 天根 |
MG1655(DE3) | 基本菌株 | 本研究 |
MG1655-∆pfkA | 来源于MG1655(DE3),pfkA缺失突变株 | 本研究 |
MG1655-∆pfkB | 来源于MG1655(DE3),pfkB缺失突变株 | 本研究 |
MG1655-∆zwf | 来源于MG1655(DE3),zwf缺失突变株 | 本研究 |
MG1655-∆ABE∷glpK | 来源于MG1655(DE3),nagABE基因位点替换为glpK的突变株 | 本研究 |
质粒 Plasmid | ||
pRed_cas9_recA_poxb300 | Cas9供体质粒 | MolecularCloud |
p∆pfkA | pfkA同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统 | 本研究 |
pRA∆pfkA | pfkA敲除质粒,用于单质粒CRISPR-Cas9系统 | 本研究 |
p∆pfkB | pfkB同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统 | 本研究 |
pRA∆pfkB | pfkB敲除质粒,用于单质粒CRISPR-Cas9系统 | 本研究 |
p∆zwf | zwf同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统 | 本研究 |
pRA∆zwf | zwf敲除质粒,用于单质粒CRISPR-Cas9系统 | 本研究 |
p∆ABE∷glpK | nagABE同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统 | 本研究 |
pRA∆ABE∷glpK | nagABE敲除质粒,用于单质粒CRISPR-Cas9系统 | 本研究 |
说明Instruction | 引物Primer | 寡核苷酸序列 Oligonucleotide sequence(5'-3') |
---|---|---|
双质粒pfkA基因敲除pfkA gene knockout(double plasmid system) | ||
gRNA扩增 gRNA amplification | pfkA-gRNA-F | TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGTGTCTGACAT-GATCAACCGGTTTTAGAGCTAGAAATAGCAAG |
gRNA-R | TCGGATCCACTAGTAACCATC | |
上游同源臂扩增Upstream homology arm amplification | pfkA-N-F | AATCACTAGTGAATTCGCGGCCGTCGGCATCTATATTTTATATAGCG |
pfkA-N-R | GAAATCAGACTACCTCTGAACTTTGGAATGCAAAATGAAATCTGTTGC | |
下游同源臂扩增Downstream homology arm amplification | pfkA-C-F | TCCAAAGTTCAGAGGTAGTCTGATTTCGGAAAAAGGCAGATTC |
pfkA-C-R | ATGCATCCAACGCGTTGGGAGCTCTCCCACCGTGTGACTGACGAATC | |
单质粒pfkA基因敲除pfkA gene knockout(single plasmid system) | ||
pfkA-F1 | GATCTGTGTCTGACATGATCAACCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG | |
pfkA-R1 | TATATAAAATATAGATGCCGAGGTCCCGCTTTGTTACAGAATGCTT | |
pfkA-up-F1 | CGGGACCTCGGCATCTATATTTTATATAGCGCGTTACGCATGG | |
pfkA-down-R1 | CGTTTTTGTCATGGCTTTCACCGTGTGACTGACG | |
pfkA-F2 | TGAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTAT | |
pfkA-R2 | CGGTTGATCATGTCAGACACAGATCTGACTCCATAACAGAGTACTCGC | |
双质粒pfkB基因敲除pfkB gene knockout(double plasmid system) | ||
gRNA扩增 gRNA amplification | pfkB-gRNA-F | TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCACGTACATGTGGAAGCAAG-GTTTTAGAGCTAGAAATAGCAAG |
gRNA-R | TCGGATCCACTAGTAACCATC | |
上游同源臂扩增Upstream homology arm amplification | pfkB-N-F | CAATCACTAGTGAATTCGCGGCCGCAGCGACCAGGCAGTGGTGTGTC |
pfkB-N-R | GGGGAATGTTTTTGCATTTCCTCCTATAGGCTGA | |
下游同源臂扩增Downstream homology arm amplification | pfkB-C-F | GGAGGAAATGCAAAAACATTCCCCCAGCATTGGGGGAATCATCAC |
pfkB-C-R | TGCATCCAACGCGTTGGGAGCTCTCCTCAAAAGACGACTGATTGCCTGC | |
单质粒pfkB基因敲除pfkB gene knockout(single plasmid system) | ||
pfkB-F1 | AGATCTCACGTACATGTGGAAGCAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG | |
pfkB-R1 | ACACCACTGCCTGGTCGCTGGGTCCCGCTTTGTTACAGAATGCTTTTAAT | |
pfkB-up-F1 | AAAGCGGGACCCAGCGACCAGGCAGTGGTGTGTCCATACCAGGTCAT | |
pfkB-down-R1 | GCGTTTTTGTCATGGCTTTTCAAAAGACGACTGATTGCCTGCCACAT | |
pfkB-F2 | TTGAAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCA | |
pfkB-R2 | CTTGCTTCCACATGTACGTGAGATCTGACTCCATAACAGAGTACTCG | |
双质粒zwf基因敲除zwf gene knockout(double plasmid system) | ||
gRNA扩增 gRNA amplification | zwf-gRNA-F | TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGCGTGCTGACTGGGATA- AAGGTTTTAGAGCTAGAAATAGCAAG |
gRNA-R | TCGGATCCACTAGTAACCATC | |
上游同源臂扩增Upstream homology arm amplification | zwf-N-F1 | AATCACTAGTGAATTCGCGGCCGCGTGTCCATGCTGCGACAGAAACG |
zwf-N-R1 | GCAGATAGTCATTCTCCTTAAGTTAACTAACCCGGTACTTAAGC | |
下游同源臂扩增Downstream homology arm amplification | zwf-C-F1 | AGTTAACTTAAGGAGAATGACTATCTGCGCTTATCCTTTATGG |
zwf-C-R1 | TCCAACGCGTTGGGAGCTCTCCCGCATTCGCTTCATGCAGGGCTTTAC | |
单质粒zwf基因敲除zwf gene knockout(single plasmid system) | ||
zwf-F1 | ATGGAGTCAGATCTGCGTGCTGACTGGGATAAAGGTTTTAG- AGCTAGAAATAGCAAGTTAAAATAAGG | |
zwf-R1 | TCTGTCGCAGCATGGACACGGGTCCCGCTTTGTTACAGAATGCT | |
zwf -up-F1 | AAGCGGGACCCGTGTCCATGCTGCGACAGAAACGATTCACCGTCG | |
zwf -down-R1 | GTTTTTGTCATGGCTTTCGCATTCGCTTCATGCAGGGCTTTACGAAT | |
zwf -F2 | ATGAAGCGAATGCGAAAGCCATGACAAAAACGCGTAACAAAAGTG | |
zwf -R2 | CTTTATCCCAGTCAGCACGCAGATCTGACTCCATAACAGAGTACTCGCCTAT | |
双质粒glpK基因与nagABE替换nagABE replacement by glpK gene(double plasmid system) | ||
gRNA扩增 gRNA amplification | nagABE-gRNA-F | TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCCTGCAGCGCCAG- TGCTTTCGTTTTAGAGCTAGAAATAGCAAG |
gRNA-R | TCGGATCCACTAGTAACCATC | |
上游同源臂扩增Upstream homology arm amplification | nagABE-N-F | AATCACTAGTGAATTCGCGGCCGAGTTTTCTACCTGAATATGCGGC |
nagABE-N-R | CTTATTGAGGTGAATAATGACACCAGGCGGACAAGCTCAGATAGG | |
下游同源臂扩增Downstream homology arm amplification | nagABE-C-F | TTGGTGACAAAACTCACAATCTGCTTTATGCCTGATGCGACGC |
nagABE-C-R | TGCATCCAACGCGTTGGGAGCTCTCCGGAGCTGATCAAAATAATCGG | |
nagB启动子扩增nagB promotor amplification | nagB-Pm-F | TGAGCTTGTCCGCCTGGTGTCATTATTCACCTCAATAAGTAAAATGTA |
nagB-Pm-R | CAAGCGTCGCATCAGGCATAAAGCAGATTGTGAGTTTTGTCACC | |
T7启动子扩增 T7 promotor amplification | nagABE-T7-F | TTTGGTGACAAAACTCACAAGGATCGAGATCTCGATCCCGCGAA |
nagABE-T7-R | GCAACGATATATTTTTTTTCAGTCATGGTATATCTCCTTCTTAAAGTT | |
glpK基因扩增 glpK gene amplification | pichi-glpk-F | CTTTAAGAAGGAGATATACCATGGGAAAAGACTATACACCAC |
pichi-glpk-R | CGTCGCATCAGGCATAAAGCAGATTAAGCAGTGTCCTTAAGCCAGCCC | |
单质粒glpK基因整合glpK gene integration(single plasmid system) | ||
ABE-F1 | GTCAGATCTCCTGCAGCGCCAGTGCTTTCGTTTTAGAGCTAGAAATAGCAAGTTAAAA- TAAGG | |
ABE -R1 | GCATATTCAGGTAGAAAACTGGTCCCGCTTTGTTACAGAATGC | |
ABE-up-F1 | GCGGGACCAGTTTTCTACCTGAATATGCGGCATGTAATGAATTTTGCCGCTGTC | |
ABE-down-R1 | TGTTACGCGTTTTTGTCATGGCTTTGGAGCTGATCAAAATAATCGGAG | |
ABE-F2 | CTCCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGC | |
ABE-R2 | CGAAAGCACTGGCGCTGCAGGAGATCTGACTCCATAACAGAGTACTC | |
突变株检测引物Detection primer for mutants | ||
zwf-JC-F | ACGGCACAAACACCGCAGGC | |
zwf-JC-R | ACATGATCAAGCGTTGCCATTGC | |
A-JC-F | TTATCCTTGTCTCGTTTCAGCGTTGGGTGGTGCG | |
A-JC-R | GTTTGATCCACTCTTTATCAATC | |
B-JC-F | CATCGCGCTCTCGATAGCCGTTAT | |
B-JC-R | GCGCAAATGCCATGCGGCATGGA | |
ABE-JC-R | TTCAACGCTGCGGTCGCGGTAC | |
ABE-JC-F | GGTTCCGCGATGGACACGCACC |
Table 2 Primers used in this study
说明Instruction | 引物Primer | 寡核苷酸序列 Oligonucleotide sequence(5'-3') |
---|---|---|
双质粒pfkA基因敲除pfkA gene knockout(double plasmid system) | ||
gRNA扩增 gRNA amplification | pfkA-gRNA-F | TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGTGTCTGACAT-GATCAACCGGTTTTAGAGCTAGAAATAGCAAG |
gRNA-R | TCGGATCCACTAGTAACCATC | |
上游同源臂扩增Upstream homology arm amplification | pfkA-N-F | AATCACTAGTGAATTCGCGGCCGTCGGCATCTATATTTTATATAGCG |
pfkA-N-R | GAAATCAGACTACCTCTGAACTTTGGAATGCAAAATGAAATCTGTTGC | |
下游同源臂扩增Downstream homology arm amplification | pfkA-C-F | TCCAAAGTTCAGAGGTAGTCTGATTTCGGAAAAAGGCAGATTC |
pfkA-C-R | ATGCATCCAACGCGTTGGGAGCTCTCCCACCGTGTGACTGACGAATC | |
单质粒pfkA基因敲除pfkA gene knockout(single plasmid system) | ||
pfkA-F1 | GATCTGTGTCTGACATGATCAACCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG | |
pfkA-R1 | TATATAAAATATAGATGCCGAGGTCCCGCTTTGTTACAGAATGCTT | |
pfkA-up-F1 | CGGGACCTCGGCATCTATATTTTATATAGCGCGTTACGCATGG | |
pfkA-down-R1 | CGTTTTTGTCATGGCTTTCACCGTGTGACTGACG | |
pfkA-F2 | TGAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTAT | |
pfkA-R2 | CGGTTGATCATGTCAGACACAGATCTGACTCCATAACAGAGTACTCGC | |
双质粒pfkB基因敲除pfkB gene knockout(double plasmid system) | ||
gRNA扩增 gRNA amplification | pfkB-gRNA-F | TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCACGTACATGTGGAAGCAAG-GTTTTAGAGCTAGAAATAGCAAG |
gRNA-R | TCGGATCCACTAGTAACCATC | |
上游同源臂扩增Upstream homology arm amplification | pfkB-N-F | CAATCACTAGTGAATTCGCGGCCGCAGCGACCAGGCAGTGGTGTGTC |
pfkB-N-R | GGGGAATGTTTTTGCATTTCCTCCTATAGGCTGA | |
下游同源臂扩增Downstream homology arm amplification | pfkB-C-F | GGAGGAAATGCAAAAACATTCCCCCAGCATTGGGGGAATCATCAC |
pfkB-C-R | TGCATCCAACGCGTTGGGAGCTCTCCTCAAAAGACGACTGATTGCCTGC | |
单质粒pfkB基因敲除pfkB gene knockout(single plasmid system) | ||
pfkB-F1 | AGATCTCACGTACATGTGGAAGCAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG | |
pfkB-R1 | ACACCACTGCCTGGTCGCTGGGTCCCGCTTTGTTACAGAATGCTTTTAAT | |
pfkB-up-F1 | AAAGCGGGACCCAGCGACCAGGCAGTGGTGTGTCCATACCAGGTCAT | |
pfkB-down-R1 | GCGTTTTTGTCATGGCTTTTCAAAAGACGACTGATTGCCTGCCACAT | |
pfkB-F2 | TTGAAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCA | |
pfkB-R2 | CTTGCTTCCACATGTACGTGAGATCTGACTCCATAACAGAGTACTCG | |
双质粒zwf基因敲除zwf gene knockout(double plasmid system) | ||
gRNA扩增 gRNA amplification | zwf-gRNA-F | TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGCGTGCTGACTGGGATA- AAGGTTTTAGAGCTAGAAATAGCAAG |
gRNA-R | TCGGATCCACTAGTAACCATC | |
上游同源臂扩增Upstream homology arm amplification | zwf-N-F1 | AATCACTAGTGAATTCGCGGCCGCGTGTCCATGCTGCGACAGAAACG |
zwf-N-R1 | GCAGATAGTCATTCTCCTTAAGTTAACTAACCCGGTACTTAAGC | |
下游同源臂扩增Downstream homology arm amplification | zwf-C-F1 | AGTTAACTTAAGGAGAATGACTATCTGCGCTTATCCTTTATGG |
zwf-C-R1 | TCCAACGCGTTGGGAGCTCTCCCGCATTCGCTTCATGCAGGGCTTTAC | |
单质粒zwf基因敲除zwf gene knockout(single plasmid system) | ||
zwf-F1 | ATGGAGTCAGATCTGCGTGCTGACTGGGATAAAGGTTTTAG- AGCTAGAAATAGCAAGTTAAAATAAGG | |
zwf-R1 | TCTGTCGCAGCATGGACACGGGTCCCGCTTTGTTACAGAATGCT | |
zwf -up-F1 | AAGCGGGACCCGTGTCCATGCTGCGACAGAAACGATTCACCGTCG | |
zwf -down-R1 | GTTTTTGTCATGGCTTTCGCATTCGCTTCATGCAGGGCTTTACGAAT | |
zwf -F2 | ATGAAGCGAATGCGAAAGCCATGACAAAAACGCGTAACAAAAGTG | |
zwf -R2 | CTTTATCCCAGTCAGCACGCAGATCTGACTCCATAACAGAGTACTCGCCTAT | |
双质粒glpK基因与nagABE替换nagABE replacement by glpK gene(double plasmid system) | ||
gRNA扩增 gRNA amplification | nagABE-gRNA-F | TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCCTGCAGCGCCAG- TGCTTTCGTTTTAGAGCTAGAAATAGCAAG |
gRNA-R | TCGGATCCACTAGTAACCATC | |
上游同源臂扩增Upstream homology arm amplification | nagABE-N-F | AATCACTAGTGAATTCGCGGCCGAGTTTTCTACCTGAATATGCGGC |
nagABE-N-R | CTTATTGAGGTGAATAATGACACCAGGCGGACAAGCTCAGATAGG | |
下游同源臂扩增Downstream homology arm amplification | nagABE-C-F | TTGGTGACAAAACTCACAATCTGCTTTATGCCTGATGCGACGC |
nagABE-C-R | TGCATCCAACGCGTTGGGAGCTCTCCGGAGCTGATCAAAATAATCGG | |
nagB启动子扩增nagB promotor amplification | nagB-Pm-F | TGAGCTTGTCCGCCTGGTGTCATTATTCACCTCAATAAGTAAAATGTA |
nagB-Pm-R | CAAGCGTCGCATCAGGCATAAAGCAGATTGTGAGTTTTGTCACC | |
T7启动子扩增 T7 promotor amplification | nagABE-T7-F | TTTGGTGACAAAACTCACAAGGATCGAGATCTCGATCCCGCGAA |
nagABE-T7-R | GCAACGATATATTTTTTTTCAGTCATGGTATATCTCCTTCTTAAAGTT | |
glpK基因扩增 glpK gene amplification | pichi-glpk-F | CTTTAAGAAGGAGATATACCATGGGAAAAGACTATACACCAC |
pichi-glpk-R | CGTCGCATCAGGCATAAAGCAGATTAAGCAGTGTCCTTAAGCCAGCCC | |
单质粒glpK基因整合glpK gene integration(single plasmid system) | ||
ABE-F1 | GTCAGATCTCCTGCAGCGCCAGTGCTTTCGTTTTAGAGCTAGAAATAGCAAGTTAAAA- TAAGG | |
ABE -R1 | GCATATTCAGGTAGAAAACTGGTCCCGCTTTGTTACAGAATGC | |
ABE-up-F1 | GCGGGACCAGTTTTCTACCTGAATATGCGGCATGTAATGAATTTTGCCGCTGTC | |
ABE-down-R1 | TGTTACGCGTTTTTGTCATGGCTTTGGAGCTGATCAAAATAATCGGAG | |
ABE-F2 | CTCCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGC | |
ABE-R2 | CGAAAGCACTGGCGCTGCAGGAGATCTGACTCCATAACAGAGTACTC | |
突变株检测引物Detection primer for mutants | ||
zwf-JC-F | ACGGCACAAACACCGCAGGC | |
zwf-JC-R | ACATGATCAAGCGTTGCCATTGC | |
A-JC-F | TTATCCTTGTCTCGTTTCAGCGTTGGGTGGTGCG | |
A-JC-R | GTTTGATCCACTCTTTATCAATC | |
B-JC-F | CATCGCGCTCTCGATAGCCGTTAT | |
B-JC-R | GCGCAAATGCCATGCGGCATGGA | |
ABE-JC-R | TTCAACGCTGCGGTCGCGGTAC | |
ABE-JC-F | GGTTCCGCGATGGACACGCACC |
基因编辑位点 Gene editing site | 单质粒CRISPR-Cas9系统编辑效率 Gene editing efficiency of the single-plasmid CRISPR-Cas9 system/% | 双质粒CRISPR-Cas9系统编辑效率 Gene editing efficiency of the double-plasmid CRISPR-Cas9 system/% |
---|---|---|
pfkA | 6 | 37 |
pfkB | 0 | 6 |
zwf | 37 | 100 |
nagABE(glpK Integration) | 0 | 10 |
Table 3 Comparison of gene editing efficiency
基因编辑位点 Gene editing site | 单质粒CRISPR-Cas9系统编辑效率 Gene editing efficiency of the single-plasmid CRISPR-Cas9 system/% | 双质粒CRISPR-Cas9系统编辑效率 Gene editing efficiency of the double-plasmid CRISPR-Cas9 system/% |
---|---|---|
pfkA | 6 | 37 |
pfkB | 0 | 6 |
zwf | 37 | 100 |
nagABE(glpK Integration) | 0 | 10 |
Fig. 2 Identification of pfkA and pfkB deletion by gel electrophoresis A:pfkA knockout using single-plasmid CRISPR-Cas9 system. B:pfkB knockout using single-plasmid CRISPR-Cas9 system. C:pfkA knockout using double-plasmid CRISPR-Cas9 system. D:pfkB knockout using double-plasmid CRISPR-Cas9 system. The target band size of the pfkA deleted strain is 1 327 bp,and the related band size of the wild-type strain is 2 290 bp. The target band size of the pfkB deleted strain is 1 287 bp,and the related band size of wild-type strain is 2 217 bp
Fig. 3 Verification of the phenotypes of pfkA and pfkB deleted mutants A:Effect of pfkA or pfkB knockout on glucose metabolism. B:Effect of pfkA or pfkB knockout on cell growth
Fig. 4 Identification of zwf deletion by gel electrophoresis A:zwf knockout using single-plasmid CRISPR-Cas9 system. B:zwf knockout using double-plasmid CRISPR-Cas9 system. The target band size of the zwf deleted strain is 1 429 bp,and the related band size of the wild-type strain is 2 905 bp
Fig. 5 Characterization of the phenotypes of zwf deleted mutant A:Effect of zwf knockout on glucose metabolism. B:Effect of zwf knockout on cell growth
Fig. 6 Identification of nagABE replaced by glpK via gel electrophoresis A:nagABE replaced by glpK gene using single-plasmid CRISPR-Cas9 system. B:nagABE replaced by glpK gene using double-plasmid CRISPR-Cas9 system. The target band size of the glpK integrated strain is 3 317 bp,and the related band size of the wild-type strain is 5 626 bp
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