inPOSE: A Flexible Toolbox for Chromosomal Cloning and Amplification of Bacterial Transgenes

• DOI: 10.3390/microorganisms10020236
• Publication Date: 2022-01-24T01:34:40Z
• Authors: Ranti Dev Shukla; Ágnes Zvara; Ákos Avramucz; Alona Yu. Biketova; Akos Nyerges; László G. Puskás; Tamás Fehér
• Keywords: Transposase; Transposition (logic); Selectable marker; Cloning (programming); Bacterial artificial chromosome; Insertion sequence

Cloning the genes and operons encoding heterologous functions in bacterial hosts is now almost exclusively carried out using plasmid vectors. This has multiple drawbacks, including need for constant selection variation copy numbers. The chromosomal integration of transgenes always offered a viable alternative; however, to date, it been limited use due its tedious nature often being single copy. We introduce here strategy that uses insertion sequences, which are simplest autonomous transposable elements insert amplify genetic cargo into chromosome. Transgene can take place either as transposition or homologous recombination, number amplification achieved controlled copy-paste transposition. display successful IS1 IS3 this purpose Escherichia coli cells various markers. demonstrate selectable genes, an unselectable gene five-gene operon up two copies step. continue with inserted cassette double-digit numbers within rounds transposase induction selection. Finally, we analyze stability cloned constructs lack find be superior all investigated plasmid-based systems. Due ubiquitous elements, believe proper design, adapted numerous other species.