Open AccessSEVAtile: a standardised DNA assembly method optimised for Pseudomonas
Author(s) -
Lammens EvelineMarie,
Boon Maarten,
Grimon Dennis,
Briers Yves,
Lavigne Rob
Publication year - 2022
Publication title -
microbial biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.287
H-Index - 74
ISSN - 1751-7915
DOI - 10.1111/1751-7915.13922
Subject(s) - pseudomonas putida , synthetic biology , computational biology , vector (molecular biology) , pseudomonas aeruginosa , dna , computer science , biology , data mining , biochemical engineering , engineering , gene , genetics , bacteria , recombinant dna
Summary 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.