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 inves-tigated 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 degrees C) is established as a reversible and switchable system, which paves the way toward novel and versatile chemicals production.

Automated summary

This study explored the temperature effect on plasmid replication in Escherichia coli and developed a thermal switchable and reversible protein expression system based on the observation. It was found that the constitutive J23100 and T7 promoter can be remodeled as a robust temperature-control system for reporter genes and functional proteins.