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. 2021 Jul 9;9(7):1476. doi: 10.3390/microorganisms9071476

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© 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Construction of double-crossover men-gene replacement strains of L. lactis NZ9000 and subsequent Cre-lox-mediated selectable marker removal result in the L. lactis deletion strains (NZ9000-Δmen). After transformation with either of the pNZ5319 knockout constructs, double crossover events result in the replacement of the target gene (men: menA, menB, menE, or menG) by the lox66-P32-cm-lox71 cassette. The cm-cassette is later removed by Cre-mediated recombination, resulting in non-functional lox72 sites.