Host-Dependent Improvement of GFP Expression in Pseudomonas putida KT2440 Using Terminators of Metagenomic Origin

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 but not in Escherichia coli DH10B. Our results suggest that replacing the default set of terminators present in the SEVA specification may be an useful strategy for fine-tuning circuit expression in P. putida, which can be leveraged for the development of new devices with improved performance in this microbial host.