铜和镍响应的双组分基因诱导表达系统的设计与优化

Design and optimization of a two-component inducible gene expression system responsive to copper and nickel

摘要:

双组分系统是一种广泛存在于细菌中的信号传递系统，能够有效地发挥信号识别、信号传导、基因调节这3种功能，从而实现信号的跨膜传输和放大。基于双组分系统构建的基因表达调控工具已被广泛应用于合成生物学和环境监测领域。传统基因诱导系统T7和P_BAD系统存在成本高、诱导剂易被细胞利用、诱导剂浓度与基因表达水平线性差等问题。为了开发低成本、诱导剂浓度与基因表达水平线性高的诱导系统，本研究基于双组分系统CusS-CusR和嵌合NrsS/CusS-CusR，成功开发了分别由Cu²⁺和Ni²⁺诱导的基因表达系统。通过优化组氨酸激酶(CusS或NrsS/CusS)与响应调节蛋白(CusR)的表达水平，将Cu²⁺诱导表达系统本底表达荧光强度由2 400 a.u.降低至852 a.u.，动态范围由1.2倍提升至8.7倍。同样结构的Ni²⁺诱导表达系统本底荧光强度为2 711 a.u.，动态范围为5.6倍。进一步通过提升目标基因的核糖体结合位点(ribosome binding site, RBS)强度和报告质粒拷贝数，使Cu²⁺诱导系统和Ni²⁺诱导系统的动态范围分别提升至50.0倍和14.3倍。与传统T7和P_BAD诱导系统相比，本研究构建的Cu²⁺和Ni²⁺诱导系统表现出更优的诱导梯度和更低的诱导成本，不仅为传统表达系统提供了有力补充，也为满足多样化实验需求提供了更多选择。

Abstract:

Two-component systems (TCS) are signal transduction systems ubiquitous in bacteria, effectively performing signal recognition, transduction, and gene regulation to achieve transmembrane signal transmission and amplification. Gene expression regulation tools based on TCS have been extensively applied in synthetic biology and environmental monitoring. The traditional gene induction systems, T7 and P_BAD systems, have problems such as high cost, ease of inducer utilisation by cells, and poor linearity between inducer concentration and gene expression level. In order to develop low-cost induction systems with high linearity between inducer concentration and gene expression level, in this study, we developed two gene expression systems induced by Cu²⁺ and Ni²⁺ based on the CusS-CusR TCS and the chimeric NrsS/CusS-CusR TCS. By optimizing the expression levels of histidine kinase (CusS or NrsS/CusS) and the response regulator (CusR), we reduced the background fluorescence intensity of the Cu²⁺-inducible system from 2 400 a.u. to 852 a.u. and improved its dynamic range from 1.2 folds to 8.7 folds. The Ni²⁺-inducible system with a similar structure demonstrated a background fluorescence intensity of 2 711 a.u. and a dynamic range of 5.6 folds. Subsequently, we increased the ribosome binding site (RBS) strength and plasmid copy number, increasing dynamic ranges of 50.0 folds and 14.3 folds for the Cu²⁺- and Ni²⁺-inducible systems, respectively. Compared with T7- and P_BAD-inducible systems, the Cu²⁺- and Ni²⁺-inducible systems developed in this study exhibit improved induction gradients and decreased induction costs, providing robust complements to existing expression systems and offering versatile options for diverse experimental applications.