Exploring temperature-mediated plasmid replication as a reversible and switchable protein expression system in genetic Escherichia coli

Background: Temperature-mediated control of gene expression has drawn extensive attention as a non-invasive approach for synthetic biology. However, the temperature-inducibility was attributed to the repressor and regulators, while temperature control on plasmid replication was rarely explored. In this study, we first investigated temperature effect on the plasmid replication in Escherichia coli. Based on the observation, a thermal switchable and reversible protein expression system was developed. Methods: The temperature effect on plasmids with 5 different replication origins was explored by measuring the fluorescent intensity, plasmid copy number (PCN) and biomass. To develop a thermal switchable and reversible platform, a proof-of-concept study was demonstrated by thermal control of the sfGFP and RFP expression with dual plasmids. Finally, aconitase and cis-aconitate decarboxylase were applied to facilitate IA production as a proof-of-function. Significant findings: The plasmid replication was controlled by temperature with positive or negative correlation, denoted “temperature-mediated plasmid replication”. Both constitutive J23100 and T7 promoter can be remodeled as a robust temperature-control system for reporter genes and functional proteins. The optimal replication origin modulating from low to high temperature (i.e., 22 to 42 °C) is established as a “reversible and switchable” system, which paves the way toward novel and versatile chemicals production.