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T7 phage-assisted evolution of riboswitches using error-prone replication and dual selection
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Goicoechea Serrano, Eduardo, Blázquez-Bondia, Carlos and Jaramillo, Alfonso (2024) T7 phage-assisted evolution of riboswitches using error-prone replication and dual selection. Scientific Reports, 14 (1). 2377. doi:10.1038/s41598-024-52049-9 ISSN 2045-2322.
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WRAP-T7-phage-assisted-evolution-riboswitches-using-error-prone-replication-dual-selection-2024.pdf - Published Version - Requires a PDF viewer. Available under License Creative Commons Attribution 4.0. Download (3769Kb) | Preview |
Official URL: https://doi.org/10.1038/s41598-024-52049-9
Abstract
Leveraging riboswitches, non-coding mRNA fragments pivotal to gene regulation, poses a challenge in effectively selecting and enriching these functional genetic sensors, which can toggle between ON and OFF states in response to their cognate inducers. Here, we show our engineered phage T7, enabling the evolution of a theophylline riboswitch. We have replaced T7’s DNA polymerase with a transcription factor controlled by a theophylline riboswitch and have created two types of host environments to propagate the engineered phage. Both types host an error-prone T7 DNA polymerase regulated by a T7 promoter along with another critical gene—either cmk or pifA, depending on the host type. The cmk gene is necessary for T7 replication and is used in the first host type for selection in the riboswitch's ON state. Conversely, the second host type incorporates the pifA gene, leading to abortive T7 infections and used for selection in the riboswitch’s OFF state. This dual-selection system, termed T7AE, was then applied to a library of 65,536 engineered T7 phages, each carrying randomized riboswitch variants. Through successive passage in both host types with and without theophylline, we observed an enrichment of phages encoding functional riboswitches that conferred a fitness advantage to the phage in both hosts. The T7AE technique thereby opens new pathways for the evolution and advancement of gene switches, including non-coding RNA-based switches, setting the stage for significant strides in synthetic biology.
Item Type: | Journal Article | |||||||||||||||||||||
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Subjects: | Q Science > QP Physiology Q Science > QR Microbiology > QR355 Virology |
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | |||||||||||||||||||||
SWORD Depositor: | Library Publications Router | |||||||||||||||||||||
Library of Congress Subject Headings (LCSH): | Riboswitches, Non-coding RNA, Genetic regulation, Bacteriophages -- Genetics, Transcription factors | |||||||||||||||||||||
Journal or Publication Title: | Scientific Reports | |||||||||||||||||||||
Publisher: | Nature Publishing Group | |||||||||||||||||||||
ISSN: | 2045-2322 | |||||||||||||||||||||
Official Date: | 29 January 2024 | |||||||||||||||||||||
Dates: |
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Volume: | 14 | |||||||||||||||||||||
Number: | 1 | |||||||||||||||||||||
Article Number: | 2377 | |||||||||||||||||||||
DOI: | 10.1038/s41598-024-52049-9 | |||||||||||||||||||||
Status: | Peer Reviewed | |||||||||||||||||||||
Publication Status: | Published | |||||||||||||||||||||
Access rights to Published version: | Open Access (Creative Commons) | |||||||||||||||||||||
Date of first compliant deposit: | 31 January 2024 | |||||||||||||||||||||
Date of first compliant Open Access: | 1 February 2024 | |||||||||||||||||||||
RIOXX Funder/Project Grant: |
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