Developing a Synthetic Biology Toolkit for Comamonas testosteroni, an Emerging Cellular Chassis for Bioremediation

Qiang Tang, Ting Lu, Shuang Jiang Liu

Research output: Contribution to journalArticle

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 languageEnglish (US)
Pages (from-to)1753-1762
Number of pages10
JournalACS synthetic biology
Volume7
Issue number7
DOIs
StatePublished - 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)

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