TY - JOUR
T1 - Developing a Synthetic Biology Toolkit for Comamonas testosteroni, an Emerging Cellular Chassis for Bioremediation
AU - Tang, Qiang
AU - Lu, Ting
AU - Liu, Shuang Jiang
N1 - We thank Dr. Zhao Tong for aid of performing FACS screening, and Dr. Howard Gelberg for editing the manuscript. This work was supported by National Natural Science Foundation of China (NSFC grant No. 31230003) to S.-J. Liu and CAS/SAFEA international partnership program to S.-J. Liu and T. Lu. This work was also supported in part by the National Science Foundation (No. 1553649 and 1227034) to T. Lu.
PY - 2018/7/20
Y1 - 2018/7/20
N2 - 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.
AB - 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.
KW - Comamonas testosteroni
KW - P promoter library
KW - T7 RNA polymerase-P system
KW - inducible promoter
KW - shuttle vehicle
KW - synthetic biology toolkit
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U2 - 10.1021/acssynbio.7b00430
DO - 10.1021/acssynbio.7b00430
M3 - Article
C2 - 29860823
AN - SCOPUS:85048128443
SN - 2161-5063
VL - 7
SP - 1753
EP - 1762
JO - ACS synthetic biology
JF - ACS synthetic biology
IS - 7
ER -