Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression
ABSTRACT Riboswitches are promising regulatory tools in synthetic biology. To date, 25 theophylline riboswitches have been developed for regulation of gene expression in bacteria. However, no one has systematically evaluated their regulatory effects. To promote efficient selection and application of...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-02-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02752-22 |
| _version_ | 1811164374824386560 |
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| author | Xun Wang Can Fang Yifei Wang Xinyu Shi Fan Yu Jin Xiong Shan-Ho Chou Jin He |
| author_facet | Xun Wang Can Fang Yifei Wang Xinyu Shi Fan Yu Jin Xiong Shan-Ho Chou Jin He |
| author_sort | Xun Wang |
| collection | DOAJ |
| description | ABSTRACT Riboswitches are promising regulatory tools in synthetic biology. To date, 25 theophylline riboswitches have been developed for regulation of gene expression in bacteria. However, no one has systematically evaluated their regulatory effects. To promote efficient selection and application of theophylline riboswitches, we examined 25 theophylline riboswitches in Escherichia coli MG1655 and found that they varied widely in terms of activation/repression ratios and expression levels in the absence of theophylline. Of the 20 riboswitches that activate gene expression, only one exhibited a high activation ratio (63.6-fold) and low expression level without theophylline. Furthermore, none of the five riboswitches that repress gene expression were more than 2.0-fold efficient. To obtain an effective repression system, we rationally designed a novel theophylline riboswitch to control a downstream gene or genes by premature transcription termination. This riboswitch allowed theophylline-dependent downregulation of the TurboRFP reporter in a dose- and time-dependent manner. Its performance profile exceeded those of previously described repressive theophylline riboswitches. We then introduced as the second part a RepA tag (protein degradation tag) coding sequence fused at the 5′-terminal end of the turborfp gene, which further reduced protein level, while not reducing the repressive effect of the riboswitch. By combining two tandem theophylline riboswitches with a RepA tag, we constructed a regulatory cassette that represses the expression of the gene(s) of interest at both the transcriptional and posttranslational levels. This regulatory cassette can be used to repress the expression of any gene of interest and represents a crucial step toward harnessing theophylline riboswitches and expanding the synthetic biology toolbox. IMPORTANCE A variety of gene expression regulation tools with significant regulatory effects are essential for the construction of complex gene circuits in synthetic biology. Riboswitches have received wide attention due to their unique biochemical, structural, and genetic properties. Here, we have not only systematically and precisely characterized the regulatory properties of previously developed theophylline riboswitches but also engineered a novel repressive theophylline riboswitch acting at the transcriptional level. By introducing coding sequences of a tandem riboswitch and a RepA protein degradation tag at the 5′ end of the reporter gene, we successfully constructed a simple and effective regulatory cassette for gene regulation. Our work provides useful biological components for the construction of synthetic biology gene circuits. |
| first_indexed | 2024-04-10T15:20:27Z |
| format | Article |
| id | doaj.art-68448a4c16294ed09ccf849d211bb88f |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-10T15:20:27Z |
| publishDate | 2023-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-68448a4c16294ed09ccf849d211bb88f2023-02-14T14:15:50ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-02-0111110.1128/spectrum.02752-22Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene RepressionXun Wang0Can Fang1Yifei Wang2Xinyu Shi3Fan Yu4Jin Xiong5Shan-Ho Chou6Jin He7State Key Laboratory of Agricultural Microbiology & Hubei Hongshan Laboratory, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, People’s Republic of ChinaState Key Laboratory of Agricultural Microbiology & Hubei Hongshan Laboratory, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, People’s Republic of ChinaState Key Laboratory of Agricultural Microbiology & Hubei Hongshan Laboratory, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, People’s Republic of ChinaState Key Laboratory of Agricultural Microbiology & Hubei Hongshan Laboratory, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, People’s Republic of ChinaState Key Laboratory of Agricultural Microbiology & Hubei Hongshan Laboratory, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, People’s Republic of ChinaState Key Laboratory of Agricultural Microbiology & Hubei Hongshan Laboratory, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, People’s Republic of ChinaState Key Laboratory of Agricultural Microbiology & Hubei Hongshan Laboratory, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, People’s Republic of ChinaState Key Laboratory of Agricultural Microbiology & Hubei Hongshan Laboratory, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, People’s Republic of ChinaABSTRACT Riboswitches are promising regulatory tools in synthetic biology. To date, 25 theophylline riboswitches have been developed for regulation of gene expression in bacteria. However, no one has systematically evaluated their regulatory effects. To promote efficient selection and application of theophylline riboswitches, we examined 25 theophylline riboswitches in Escherichia coli MG1655 and found that they varied widely in terms of activation/repression ratios and expression levels in the absence of theophylline. Of the 20 riboswitches that activate gene expression, only one exhibited a high activation ratio (63.6-fold) and low expression level without theophylline. Furthermore, none of the five riboswitches that repress gene expression were more than 2.0-fold efficient. To obtain an effective repression system, we rationally designed a novel theophylline riboswitch to control a downstream gene or genes by premature transcription termination. This riboswitch allowed theophylline-dependent downregulation of the TurboRFP reporter in a dose- and time-dependent manner. Its performance profile exceeded those of previously described repressive theophylline riboswitches. We then introduced as the second part a RepA tag (protein degradation tag) coding sequence fused at the 5′-terminal end of the turborfp gene, which further reduced protein level, while not reducing the repressive effect of the riboswitch. By combining two tandem theophylline riboswitches with a RepA tag, we constructed a regulatory cassette that represses the expression of the gene(s) of interest at both the transcriptional and posttranslational levels. This regulatory cassette can be used to repress the expression of any gene of interest and represents a crucial step toward harnessing theophylline riboswitches and expanding the synthetic biology toolbox. IMPORTANCE A variety of gene expression regulation tools with significant regulatory effects are essential for the construction of complex gene circuits in synthetic biology. Riboswitches have received wide attention due to their unique biochemical, structural, and genetic properties. Here, we have not only systematically and precisely characterized the regulatory properties of previously developed theophylline riboswitches but also engineered a novel repressive theophylline riboswitch acting at the transcriptional level. By introducing coding sequences of a tandem riboswitch and a RepA protein degradation tag at the 5′ end of the reporter gene, we successfully constructed a simple and effective regulatory cassette for gene regulation. Our work provides useful biological components for the construction of synthetic biology gene circuits.https://journals.asm.org/doi/10.1128/spectrum.02752-22theophylline riboswitchbiological partstranscriptional regulationposttranslational regulationprotein degradation tag |
| spellingShingle | Xun Wang Can Fang Yifei Wang Xinyu Shi Fan Yu Jin Xiong Shan-Ho Chou Jin He Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression Microbiology Spectrum theophylline riboswitch biological parts transcriptional regulation posttranslational regulation protein degradation tag |
| title | Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression |
| title_full | Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression |
| title_fullStr | Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression |
| title_full_unstemmed | Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression |
| title_short | Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression |
| title_sort | systematic comparison and rational design of theophylline riboswitches for effective gene repression |
| topic | theophylline riboswitch biological parts transcriptional regulation posttranslational regulation protein degradation tag |
| url | https://journals.asm.org/doi/10.1128/spectrum.02752-22 |
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