Journal
ACS SYNTHETIC BIOLOGY
Volume 7, Issue 7, Pages 1753-1762Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.7b00430
Keywords
synthetic biology toolkit; shuttle vehicle; inducible promoter; T7 RNA polymerase-P-T7 system; P-T7DOM promoter library; Comamonas testosteroni
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Funding
- National Natural Science Foundation of China (NSFC) [31230003]
- CAS/SAFEA international partnership program
- National Science Foundation [1553649, 1227034]
- Div Of Engineering Education and Centers
- Directorate For Engineering [1227034] Funding Source: National Science Foundation
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1553649] Funding Source: National Science Foundation
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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-P-T7 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.
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