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Using the Cre-lox system to randomize target gene expression states and generate diverse phenotypes.

ABSTRACT: Modifying the expression of multiple genes enables both deeper understanding of their function and the engineering of complex multigenic cellular phenotypes. However, deletion or overexpression of multiple genes is typically laborious and involves multiple sequential genetic modifications. Here we describe a strategy to randomize the expression state of multiple genes in Saccharomyces cerevisiae using Cre-loxP recombination. By inserting promoters flanked by inverted loxP sites in front of a gene of interest we can randomly alter its expression by turning it OFF or ON, or between four distinct expression states. We show at least 6 genes can be randomized independently and argue that using orthogonal loxP sites should increase this number to at least 15. Finally, we show how combining this strategy with mating allows easy introduction of native regulation as an additional expression state and use this to probe the role of four different enzymes involved in base excision repair in tolerance to methyl methane sulfonate (MMS), a genotoxic DNA alkylating agent. The set of vectors developed here can be used to randomize the expression of both heterologous genes and endogenous genes, and could immediately prove useful for metabolic engineering in yeast. Because Cre-loxP recombination works in many organisms, this strategy should be readily extendable.

SUBMITTER: Niesner B

PROVIDER: S-EPMC4264681 | biostudies-other | 2013 Oct

REPOSITORIES: biostudies-other

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Using the Cre-lox system to randomize target gene expression states and generate diverse phenotypes.

Niesner Bradley B Maheshri Narendra N

Biotechnology and bioengineering 20130603 10

Modifying the expression of multiple genes enables both deeper understanding of their function and the engineering of complex multigenic cellular phenotypes. However, deletion or overexpression of multiple genes is typically laborious and involves multiple sequential genetic modifications. Here we describe a strategy to randomize the expression state of multiple genes in Saccharomyces cerevisiae using Cre-loxP recombination. By inserting promoters flanked by inverted loxP sites in front of a gen ...[more]

PMID: 23733452

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Project description:The classic strategy to achieve gene deletion variants is based on double-crossover integration of nonreplicating vectors into the genome. In addition, recombination systems such as Cre-lox have been used extensively, mainly for eukaryotic organisms. This study presents the construction of a Cre-lox-based system for multiple gene deletions in Lactobacillus plantarum that could be adapted for use on gram-positive bacteria. First, an effective mutagenesis vector (pNZ5319) was constructed that allows direct cloning of blunt-end PCR products representing homologous recombination target regions. Using this mutagenesis vector, double-crossover gene replacement mutants could be readily selected based on their antibiotic resistance phenotype. In the resulting mutants, the target gene is replaced by a lox66-P(32)-cat-lox71 cassette, where lox66 and lox71 are mutant variants of loxP and P(32)-cat is a chloramphenicol resistance cassette. The lox sites serve as recognition sites for the Cre enzyme, a protein that belongs to the integrase family of site-specific recombinases. Thus, transient Cre recombinase expression in double-crossover mutants leads to recombination of the lox66-P(32)-cat-lox71 cassette into a double-mutant loxP site, called lox72, which displays strongly reduced recognition by Cre. The effectiveness of the Cre-lox-based strategy for multiple gene deletions was demonstrated by construction of both single and double gene deletions at the melA and bsh1 loci on the chromosome of the gram-positive model organism Lactobacillus plantarum WCFS1. Furthermore, the efficiency of the Cre-lox-based system in multiple gene replacements was determined by successive mutagenesis of the genetically closely linked loci melA and lacS2 in L. plantarum WCFS1. The fact that 99.4% of the clones that were analyzed had undergone correct Cre-lox resolution emphasizes the suitability of the system described here for multiple gene replacement and deletion strategies in a single genetic background.

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Using the Cre-lox system to randomize target gene expression states and generate diverse phenotypes.

Publications

Using the Cre-lox system to randomize target gene expression states and generate diverse phenotypes.

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