A termination-aware vector design improves heterologous gene expression in Pseudomonas putida

Transcriptional terminators are key players in the flow of genetic information, but are often overlooked in circuit design. In this work, we used the Standard European Vector Architecture (SEVA) as a scaffold to investigate the effects of different terminators in the output of a reporter construct expressed in two bacterial species and found that replacing the conventional T1 and T0 transcriptional terminators of the SEVA vector format with a set of broad-host metagenomic terminators resulted in a significant improvement in the signal of a fluorescent device in Pseudomonas putida KT2440. Interestingly, this new vector design displayed the same performance as pSEVA231 in Escherichia coli DH10B. Our results support the notion that transcriptional terminators may affect the interconnected mesh of biological processes taking place in gene expression, leading to a host-dependent output that should be heeded for optimal circuit expression in different microbial hosts.