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

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Developing a Synthetic Biology Toolkit for Comamonas testosteroni, an Emerging Cellular Chassis for Bioremediation
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-06-03 00:00:00 , DOI: 10.1021/acssynbio.7b00430
Qiang Tang _{1,

2} , Ting Lu , Shuang-Jiang Liu _{1,

2,

3}

Affiliation

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.

中文翻译：

为Comamonas睾丸激素（一种用于生物修复的新兴细胞底盘）开发合成生物学工具包。

合成生物学正迅速进入一个新阶段，该阶段强调环境修复等现实应用。近年来，由于其天然污染物的降解能力，睾丸激素（Comamonas testosteroni）已成为有前途的生物修复基础。然而，由于缺乏基本的基因表达工具，其应用受到了阻碍。在这里，我们介绍了一种合成生物学工具包，该工具包可快速创建睾丸丙酸杆菌中的功能基因电路。我们首先构建了一个穿梭系统，该系统可以在大肠杆菌中进行高效的电路构建，并在睾丸梭菌中进行必要的表型测试。。然后，我们测试了一组野生型诱导型启动子，并进一步使用了杂交策略来创建工程化启动子，以扩展表达强度和动力学。此外，我们测试了T7 RNA聚合酶-P _T7启动子系统，并通过启动子突变减少了其漏表达，以表达基因。通过将随机文库构建与FACS筛选相结合，我们进一步开发了合成的T7启动子文库，以赋予更广泛的表达强度和动态特性。这项研究提供了一套有价值的工具，可在睾丸假单胞菌中工程化基因回路，从而促进将生物体确立为用于生物修复目的的有用微生物底盘。

更新日期：2018-06-03

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