Fluorescence-Based Assays to Evaluate the Functionality of Phage-Derived Regulatory Elements for Applications in...

Precise regulation of gene expression is fundamental to microbial synthetic biology, to enable the construction of programmable biological systems. Promoters and terminators play a pivotal role in this regulation, yet their availability and functionality are often limited to model organisms, hindering broader application in non-model bacterial hosts. Bacteriophages, with their diverse and efficient regulatory elements, offer a promising solution. Their promoters-either host-specific or phage-specific-provide both compatibility with native transcriptional systems and orthogonality for insulated gene expression. However, functional validation remains essential. This chapter presents a fluorescence-based workflow for characterizing phage-derived promoters and terminators in vivo, offering a scalable approach to expand the synthetic biology toolkit across diverse bacterial chassis. Using the SEVAtile DNA assembly method, modular constructs are generated to test host-specific and phage-specific promoters, as well as terminators, exemplified for Pseudomonas chlororaphis. Constructs are introduced via electroporation and assessed using plate reader-based fluorescence measurements of msfGFP and mCherry reporters. Promoter strength and terminator efficiency are quantified and normalized to cell growth, enabling comparative analysis across constructs. This scalable and adaptable workflow provides a robust framework for characterizing novel regulatory elements and expanding the synthetic biology toolkit for diverse bacterial systems.