Abstract
Synthetic biology is rapidly evolving into a new phase that emphasizes real-world applications such as environmental remediation. Recently, Comamonas testosteroni has become a promising chassis for bioremediation due to its natural pollutant-degrading capacity; however, its application is hindered by the lack of fundamental gene expression tools. Here, we present a synthetic biology toolkit that enables rapid creation of functional gene circuits in C. testosteroni. We first built a shuttle system that allows efficient circuit construction in E. coli and necessary phenotypic testing in C. testosteroni. Then, we tested a set of wildtype inducible promoters, and further used a hybrid strategy to create engineered promoters to expand expression strength and dynamics. Additionally, we tested the T7 RNA Polymerase-PT7 promoter system and reduced its leaky expression through promoter mutation for gene expression. By coupling random library construction with FACS screening, we further developed a synthetic T7 promoter library to confer a wider range of expression strength and dynamic characteristics. This study provides a set of valuable tools to engineer gene circuits in C. testosteroni, facilitating the establishment of the organism as a useful microbial chassis for bioremediation purposes.
Original language | English (US) |
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Pages (from-to) | 1753-1762 |
Number of pages | 10 |
Journal | ACS synthetic biology |
Volume | 7 |
Issue number | 7 |
DOIs | |
State | Published - Jul 20 2018 |
Keywords
- Comamonas testosteroni
- P promoter library
- T7 RNA polymerase-P system
- inducible promoter
- shuttle vehicle
- synthetic biology toolkit
ASJC Scopus subject areas
- Biomedical Engineering
- Biochemistry, Genetics and Molecular Biology (miscellaneous)