Translation initiation consistency between in vivo and in vitro bacterial protein expression systems
Strict on-demand control of protein synthesis is a crucial aspect of synthetic biology. The 5′-terminal untranslated region (5′-UTR) is an essential bacterial genetic element that can be designed for the regulation of translation initiation. However, there is insufficient systematical data on the co...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-05-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1201580/full |
| _version_ | 1797820499627081728 |
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| author | Jiaojiao Li Jiaojiao Li Peixian Li Peixian Li Qian Liu Qian Liu Jinjin Li Jinjin Li Hao Qi Hao Qi Hao Qi |
| author_facet | Jiaojiao Li Jiaojiao Li Peixian Li Peixian Li Qian Liu Qian Liu Jinjin Li Jinjin Li Hao Qi Hao Qi Hao Qi |
| author_sort | Jiaojiao Li |
| collection | DOAJ |
| description | Strict on-demand control of protein synthesis is a crucial aspect of synthetic biology. The 5′-terminal untranslated region (5′-UTR) is an essential bacterial genetic element that can be designed for the regulation of translation initiation. However, there is insufficient systematical data on the consistency of 5′-UTR function among various bacterial cells and in vitro protein synthesis systems, which is crucial for the standardization and modularization of genetic elements in synthetic biology. Here, more than 400 expression cassettes comprising the GFP gene under the regulation of various 5′-UTRs were systematically characterized to evaluate the protein translation consistency in the two popular Escherichia coli strains of JM109 and BL21, as well as an in vitro protein expression system based on cell lysate. In contrast to the very strong correlation between the two cellular systems, the consistency between in vivo and in vitro protein translation was lost, whereby both in vivo and in vitro translation evidently deviated from the estimation of the standard statistical thermodynamic model. Finally, we found that the absence of nucleotide C and complex secondary structure in the 5′-UTR significantly improve the efficiency of protein translation, both in vitro and in vivo. |
| first_indexed | 2024-03-13T09:39:17Z |
| format | Article |
| id | doaj.art-29378d4d28e14eb79815e513c32e9654 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-03-13T09:39:17Z |
| publishDate | 2023-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-29378d4d28e14eb79815e513c32e96542023-05-25T04:34:29ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-05-011110.3389/fbioe.2023.12015801201580Translation initiation consistency between in vivo and in vitro bacterial protein expression systemsJiaojiao Li0Jiaojiao Li1Peixian Li2Peixian Li3Qian Liu4Qian Liu5Jinjin Li6Jinjin Li7Hao Qi8Hao Qi9Hao Qi10School of Chemical Engineering and Technology, Tianjin University, Tianjin, ChinaFrontier Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, ChinaSchool of Chemical Engineering and Technology, Tianjin University, Tianjin, ChinaFrontier Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, ChinaSchool of Chemical Engineering and Technology, Tianjin University, Tianjin, ChinaFrontier Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, ChinaSchool of Chemical Engineering and Technology, Tianjin University, Tianjin, ChinaFrontier Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, ChinaSchool of Chemical Engineering and Technology, Tianjin University, Tianjin, ChinaFrontier Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, ChinaZhejiang Shaoxing Research Institute of Tianjin University, Shaoxing, ChinaStrict on-demand control of protein synthesis is a crucial aspect of synthetic biology. The 5′-terminal untranslated region (5′-UTR) is an essential bacterial genetic element that can be designed for the regulation of translation initiation. However, there is insufficient systematical data on the consistency of 5′-UTR function among various bacterial cells and in vitro protein synthesis systems, which is crucial for the standardization and modularization of genetic elements in synthetic biology. Here, more than 400 expression cassettes comprising the GFP gene under the regulation of various 5′-UTRs were systematically characterized to evaluate the protein translation consistency in the two popular Escherichia coli strains of JM109 and BL21, as well as an in vitro protein expression system based on cell lysate. In contrast to the very strong correlation between the two cellular systems, the consistency between in vivo and in vitro protein translation was lost, whereby both in vivo and in vitro translation evidently deviated from the estimation of the standard statistical thermodynamic model. Finally, we found that the absence of nucleotide C and complex secondary structure in the 5′-UTR significantly improve the efficiency of protein translation, both in vitro and in vivo.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1201580/fullcell-free systemgene expression regulationprotein engineeringsynthetic biologyE. coli |
| spellingShingle | Jiaojiao Li Jiaojiao Li Peixian Li Peixian Li Qian Liu Qian Liu Jinjin Li Jinjin Li Hao Qi Hao Qi Hao Qi Translation initiation consistency between in vivo and in vitro bacterial protein expression systems Frontiers in Bioengineering and Biotechnology cell-free system gene expression regulation protein engineering synthetic biology E. coli |
| title | Translation initiation consistency between in vivo and in vitro bacterial protein expression systems |
| title_full | Translation initiation consistency between in vivo and in vitro bacterial protein expression systems |
| title_fullStr | Translation initiation consistency between in vivo and in vitro bacterial protein expression systems |
| title_full_unstemmed | Translation initiation consistency between in vivo and in vitro bacterial protein expression systems |
| title_short | Translation initiation consistency between in vivo and in vitro bacterial protein expression systems |
| title_sort | translation initiation consistency between in vivo and in vitro bacterial protein expression systems |
| topic | cell-free system gene expression regulation protein engineering synthetic biology E. coli |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1201580/full |
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