Chapter 16 : Genetic Tools for the Enhancement of Probiotic Properties

Schematic representation of dsDNA recombineering in L. plantarum. (A) A piece of dsDNA harboring the lox66-cat-lox71 cassette (lox66 and lox71 sites, red; Cat/CM marker, yellow) and regions with homology to the genomic insertion site (H1, green; H2, blue) is transformed into L. plantarum expressing an exonuclease (Lp0642), a possible host nuclease inhibitor (Lp0640), and an ssDNA binding protein (Lp0641). (B) Once the dsDNA fragments are integrated into the genome, which renders the cells resistant to CM (CMr), bacteria are transformed with a plasmid that induces the expression of the recombinase Cre to recombine the loxP sites. This recombination deletes the CM marker contained inside the lox sites, rendering the cells CM-sensitive and leaving a modified loxP site (lox72).

Schematic representation of ssDNA recombineering in L. reuteri. (A) An oligomer harboring the mutation (red) and regions with homology to the genomic insertion site (blue) is transformed into cells expressing RecT. (B) The oligomer is incorporated into the lagging strand being synthesized at the DNA replication fork, generating a mutant with a mixed genotype (GT). (C) Screening of the mutant is done by MAMA-PCR, using two oligomers that amplify from a WT sequence (black) and a third oligomer (red-blue) that only amplifies when the mutation is incorporated. Thus, two amplicons are generated in the case of the mutant (lane 1), whereas only one amplicon is generated in the case of the WT (lanes 2 to 5). After the mixed genotype is identified, single colony purification is done to isolate cells containing only the mutation (pure genotype).

Overview of recombineering/CRISPR-Cas9 genome engineering in L. reuteri. (A) An oligomer harboring the mutation (red) and regions with homology to the genomic insertion site (blue) is transformed into L. reuteri expressing RecT, Cas9, and its tracrRNA. (B) The oligomer targets the lagging strains and is incorporated into one of the DNA strands at the replication fork. When cells divide, a mixed population of WT and mutant cells is generated. (C) A plasmid expressing guide RNA (pgRNA) that targets the WT sequence is introduced into the cell. Cas9 is directed by the gRNA to cleave DNA of cells that did not incorporate the mutations (WT), whereas cells that incorporate the mutation are void of cleavage (mutant).