Massive integration of large gene libraries in the chromosome of Escherichia coli.

ABSTRACT: Large gene libraries are frequently created in Escherichia coli plasmids, which can induce cell toxicity and expression instability due to the high gene dosage. To address these limitations, gene libraries can be integrated in a single copy into the bacterial chromosome. Here, we describe an efficient system for the massive integration (MAIN) of large gene libraries in the E. coli chromosome that generates in-frame gene fusions that are expressed stably. MAIN uses a thermosensitive integrative plasmid that is linearized in vivo to promote extensive integration of the gene library via homologous recombination. Positive and negative selections efficiently remove bacteria lacking gene integration in the target site. We tested MAIN with a library of 10⁷ V_HH genes that enc

SUBMITTER: Cerdan L 

PROVIDER: S-EPMC10832519 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature