CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently
Abstract T7 Expression System is a common method of ensuring tight control and high-level induced expression. However, this system can only work in some bacterial strains in which the T7 RNA Polymerase gene resides in the chromosome. In this study, we successfully introduced a chromosomal copy of th...
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2021-08-01
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| Series: | Journal of Biological Engineering |
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| Online Access: | https://doi.org/10.1186/s13036-021-00270-9 |
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doaj-73fab2ce90204ee48c427f22885a3f0f2021-08-15T11:47:37ZengBMCJournal of Biological Engineering1754-16112021-08-0115111610.1186/s13036-021-00270-9CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficientlyChangchuan Ye0Xi Chen1Mengjie Yang2Xiangfang Zeng3Shiyan Qiao4State Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural UniversityState Key Laboratory for Agro-Biotechnology, and Ministry of Agriculture and Rural Affairs, Key Laboratory for Pest Monitoring and Green Management, Department of Plant Pathology, China Agricultural UniversityNational Feed Engineering Technology Research CentreState Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural UniversityState Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural UniversityAbstract T7 Expression System is a common method of ensuring tight control and high-level induced expression. However, this system can only work in some bacterial strains in which the T7 RNA Polymerase gene resides in the chromosome. In this study, we successfully introduced a chromosomal copy of the T7 RNA Polymerase gene under control of the lacUV5 promoter into Escherichia coli BW25113. The T7 Expression System worked efficiently in this mutant strain named BW25113-T7. We demonstrated that this mutant strain could satisfactorily produce 5-Aminolevulinic Acid via C5 pathway. A final study was designed to enhance the controllability of T7 Expression System in this mutant strain by constructing a T7 Promoter Variants Library. These efforts advanced E. coli BW25113-T7 to be a practical host for future metabolic engineering efforts.https://doi.org/10.1186/s13036-021-00270-9E. coliCRISPR/cas9T7 Expression SystemFluorescent Protein5-Aminolevulinic Acidpromoter variants |
| collection |
DOAJ |
| language |
English |
| format |
Article |
| sources |
DOAJ |
| author |
Changchuan Ye Xi Chen Mengjie Yang Xiangfang Zeng Shiyan Qiao |
| spellingShingle |
Changchuan Ye Xi Chen Mengjie Yang Xiangfang Zeng Shiyan Qiao CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently Journal of Biological Engineering E. coli CRISPR/cas9 T7 Expression System Fluorescent Protein 5-Aminolevulinic Acid promoter variants |
| author_facet |
Changchuan Ye Xi Chen Mengjie Yang Xiangfang Zeng Shiyan Qiao |
| author_sort |
Changchuan Ye |
| title |
CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently |
| title_short |
CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently |
| title_full |
CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently |
| title_fullStr |
CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently |
| title_full_unstemmed |
CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently |
| title_sort |
crispr/cas9 mediated t7 rna polymerase gene knock-in in e. coli bw25113 makes t7 expression system work efficiently |
| publisher |
BMC |
| series |
Journal of Biological Engineering |
| issn |
1754-1611 |
| publishDate |
2021-08-01 |
| description |
Abstract T7 Expression System is a common method of ensuring tight control and high-level induced expression. However, this system can only work in some bacterial strains in which the T7 RNA Polymerase gene resides in the chromosome. In this study, we successfully introduced a chromosomal copy of the T7 RNA Polymerase gene under control of the lacUV5 promoter into Escherichia coli BW25113. The T7 Expression System worked efficiently in this mutant strain named BW25113-T7. We demonstrated that this mutant strain could satisfactorily produce 5-Aminolevulinic Acid via C5 pathway. A final study was designed to enhance the controllability of T7 Expression System in this mutant strain by constructing a T7 Promoter Variants Library. These efforts advanced E. coli BW25113-T7 to be a practical host for future metabolic engineering efforts. |
| topic |
E. coli CRISPR/cas9 T7 Expression System Fluorescent Protein 5-Aminolevulinic Acid promoter variants |
| url |
https://doi.org/10.1186/s13036-021-00270-9 |
| work_keys_str_mv |
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