Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Biochemical Research Methods, Biochemistry & Molecular Biology, TRANSCRIPTION, EXPRESSION, CLONING, DESIGN, RANGE, RNA, Escherichia coli, Bacteriophages, Terminator Regions, Genetic, Transcription, Genetic, Pseudomonas, G096519N#54969813, 0304 Medicinal and Biomolecular Chemistry, 0601 Biochemistry and Cell Biology, 0903 Biomedical Engineering, 3101 Biochemistry and cell biology, 3102 Bioinformatics and computational biology

Abstract:

Efficient transcriptional terminators are essential for the performance of genetic circuitry in microbial SynBio hosts. In recent years, several libraries of characterized strong terminators have become available for model organisms such as Escherichia coli. Conversely, terminator libraries for nonmodel species remain scarce, and individual terminators are often ported over from model systems, leading to unpredictable performance in their new hosts. In this work, we mined the genomes of Pseudomonas infecting phages LUZ7 and LUZ100 for transcriptional terminators utilizing the full-length RNA sequencing technique "ONT-cappable-seq" and validated these terminators in three Gram-negative hosts using a terminator trap assay. Based on these results, we present nine terminators for E. coli, Pseudomonas putida, and Pseudomonas aeruginosa, which outperform current reference terminators. Among these, terminator LUZ7 T50 displays potent bidirectional activity. These data further support that bacteriophages, as evolutionary-adapted natural predators of the targeted bacteria, provide a valuable source of microbial SynBio parts.