Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Biotechnology & Applied Microbiology, Microbiology, GENE-EXPRESSION, RNA-POLYMERASE, AERUGINOSA, TRANSCRIPTION, RANGE, LYSOZYME, SYSTEM, DNA, Genetic Vectors, Plasmids, Pseudomonas, Pseudomonas putida, Reproducibility of Results, G096519N#54969813, 0605 Microbiology, 3106 Industrial biotechnology, 3107 Microbiology

Abstract:

To meet the needs of synthetic biologists, DNA assembly methods have transformed from simple ‘cut-and-paste’ procedures to highly advanced, standardised assembly techniques. Implementing these standardised DNA assembly methods in biotechnological research conducted in non-model hosts, including Pseudomonas putida and Pseudomonas aeruginosa, could greatly benefit reproducibility and predictability of experimental results. SEVAtile is a Type IIs-based assembly approach, which enables the rapid and standardised assembly of genetic parts – or tiles – to create genetic circuits in the established SEVA-vector backbone. Contrary to existing DNA assembly methods, SEVAtile is an easy and straightforward method, which is compatible with any vector, both SEVA- and non-SEVA. To prove the efficiency of the SEVAtile method, a three-vector system was successfully generated to independently co-express three different proteins in P. putida and P. aeruginosa. More specifically, one of the vectors, pBGDes, enables genomic integration of assembled circuits in the Tn7 landing site, while self-replicatory vectors pSTDesX and pSTDesR enable inducible expression from the XylS/Pm and RhaRS/PrhaB expression systems, respectively. Together, we hope these vector systems will support research in both the microbial SynBio and Pseudomonas field.