SEVAtile: a standardised DNA assembly method optimised for Pseudomonas

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.