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Characterization and Diversification of AraC/XylS Family Regulators Guided by Transposon Sequencing
Open accessPublished version of a preprint
Abstract
Abstract In this study, we explored the development of engineered inducible systems. Publicly available data from previous transposon sequencing assays were used to identify regulators of metabolism in Pseudomonas putida KT2440. For the AraC-family regulators (AFRs) represented in this data, we posited AFR/promoter/inducer groupings. Eleven promoters were characterized for a response to their proposed inducers in P. putida , and the resultant data were used to create and test nine two-plasmid sensor systems in E. coli . Several of these were further developed into a palette of single-plasmid inducible systems. From these experiments, we observed an unreported inducer response from a previously characterized AFR, demonstrated that the addition of a P. putida transporter improved the sensor dynamics of an AFR in E. coli , and identified an uncharacterized AFR with a novel potential inducer specificity. Finally, targeted mutations in an AFR, informed by structural predictions, enabled further diversification of these inducible plasmids. Graphical Abstract
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Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., et al. (2021) · Nature · doi:10.1038/s41586-021-03819-2 · Cited by 43,764
Accurate prediction of protein structures and interactions using a 3-track network
Baek, M., DiMaio, F., Anishchenko, I., Dauparas, J., Ovchinnikov, S., Lee, G. R., Wang, J., Cong, Q., Kinch, L. N., Schaeffer, R. D., et al. (2021) · doi:10.1101/2021.06.14.448402 · Cited by 5,549
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Cooper, S., Khatib, F., Treuille, A., Barbero, J., Lee, J., Beenen, M., Leaver-Fay, A., Baker, D., Popović, Z., & players, F. (2010) · Nature · doi:10.1038/nature09304 · Cited by 1,368
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Metadata
- Authors
- Pearson, A. N., Incha, M. R., Ho, C. N., Schmidt, M., Roberts, J. B., Nava, A. A., & Keasling, J. D.
- Affiliations
- Lawrence Berkeley National Laboratory (US)
- Corresponding author
- Keasling, Jay D.
- ORCID
- Venue
- ACS Synthetic Biology
- Publisher
- American Chemical Society (ACS)
- Published
- 2023-12-19
- Volume
- 13
- Issue
- 1
- Pages
- 206-219
- ISSN
- 2161-5063 (ISSN-L)
- OpenAlex
- W4389946989
- Cited by
- 5
- References
- 4
- Citations by year
- 2026: 1 · 2025: 3 · 2024: 1
- FWCI
- 0.44
- Type
- journal article
- Language
- en
- Open access
- green
- dx.doi.org
- Licence
- Locations
- ACS Synthetic Biology (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
- PubMed (publishedVersion)
- eScholarship (California Digital Library) (submittedVersion, cc-by)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- RWTH Publications (RWTH Aachen) (submittedVersion)
- Technical University of Denmark, DTU Orbit (Technical University of Denmark, DTU) (submittedVersion)
- Funders
- Biological and Environmental Research
- Division of Graduate Education (2018253421)
- Bioenergy Technologies Office (DE-AC02-05CH11231)
- Office of Science
- National Defense Science and Engineering Graduate (FA9550-21-F-0003)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Keywords
- Pseudomonas putida KT2440, transposon sequencing, promoter characterization, inducer specificity, metabolic regulation, targeted mutations, structure prediction, Escherichia coli
- MeSH terms
- Bacterial Proteins, Escherichia coli, Plasmids, Promoter Regions, Genetic, Gene Expression Regulation, Bacterial, Pseudomonas putida, Escherichia coli Proteins, AraC Transcription Factor
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