TECS: a toxin expression control strategy as a tool for optimization of inducible promoters

Transcriptional control of gene expression is a widely utilized regulatory mechanism in synthetic biology, biotechnology and recombinant protein production. It is achieved by utilization of naturally occurring promoters responding to nutrients or chemicals. Despite their regulatory properties, these promoters often possess features which diminish their utility for biotechnology. High basal expression level and low induction ratio can be removed using genetic engineering techniques, although this process is often laborious and time-consuming.

In order to facilitate optimization process for inducible promoters, we developed a simple method based on a conditional toxin expression which we abbreviate as toxin expression control strategy (TECS). In the presence of sucrose, SacB enzyme from Bacillus subtilis synthesizes levans which cause Eschericha coli cell lysis. However, in the absence of sucrose the enzyme does not affect the growth of the host. We utilized this feature to develop a two-step protocol allowing for efficient selection of inducible promoter variants. Using TECS we were able to modify the well-described p_BAD promoter to decrease its leakage while maintaining high activity upon induction. Furthermore, we used the method to test transcriptional interference of lambda phage-derived sequence and optimize it for higher induction levels through random mutagenesis.

We show that TECS is an efficient tool for optimization and development of inducible promoter systems in E. coli. Our strategy is very effective in the selection of promoter variants with improved properties. Its simplicity and short hands-on time make it an attractive method to optimize existing promoters and to construct novel, engineered genetic elements which improve properties of an inducible promoter system.