Construction of promoters with tight regulation on chromosome of Escherichia coli

Abstract:

[Background] The leakage expression of promoters is a major concern in metabolic engineering and synthetic biology. To explore promoters with tight regulation like a switch would resolve this problem. [Objective] In order to avoid the shortcomings of using plasmid as research auxiliary, the aim of this study is to construct and assess promoter with tight regulation on chromosome. [Methods] Four elements including tetO, lacO, araC and rhaR regulated by TetR, LacI, AraC and RhaR respectively, were selected for designing six promoters including PtetO2, PtetO3, PlacO2, PlacO3, PlacO+ara and PlacO+rha. CRISPR/Cas9 system was applied to integrate these promoters into chromosome of Escherichia coli ATCC 8739. Promoter strength and relative expression with or without inducing agents were determined by the fluorescent quantities of GFP protein. [Results] Hybrid promoter PlacO+rha was the best one with tight regulation. The expression strength of PlacO+rha was 0.02 in absence of the inducer, and the maximum expression intensity was 12-fold higher than that of the induced lacZ promoter, of which the range of expression level was 600-fold. [Conclusion] This study would provide a good foundation for precisely regulating gene expression in metabolic engineering and synthetic biology.