Figure 1
Overview of recombineering in L. reuteri. (i) Electrocompetent cells in which RecT is expressed are transformed with a recombineering oligonucleotide. Transformation efficiencies of >105cfu/mg DNA are required for high recombineering efficiencies. Wavy black lines with a red dot represent recombineering oligonucleotides with multiple non-complementary bases; the green circle represents a bacterial cell; pJP042 is the sakacin-based expression vector that contains recT; the black double helix represents chromosomal DNA; expressed RecT proteins are denoted in the cell. (ii) An oligonucleotide identical to the lagging strand contains multiple non-complementary bases that avoid the mismatch repair system resulting in increased recombineering efficiency. (iii) Viable cells are recovered on antibiotic-free plates and recombinants are detected by a mismatch amplification mutation assay-PCR (MAMA-PCR). Two oligonucleotides (blue) will yield a 1-kb fragment, whereas a third oligonucleotide (red) will only be extended by the polymerase when the mutations are incorporated in the chromosome yielding a second amplicon of 500 bp. As the recombineering oligonucleotide only targets one strand during DNA replication the colonies will be of mixed genotype. The red dot on the chromosome indicates that the mutations are incorporated. (iv) Single colony purification is performed to separate the wild-type genotype from the mutant genotype. (v) MAMA-PCR is repeated as described in section iii to identify a pure genotype mutant. A 1:1 ratio of wild-type and mutant genotypes is suggestive that during replication a single chromosome is being replicated per cell. (vi) The recombineering plasmid pJP042 can now be cured from the mutant strain by passaging bacteria without antibiotic selection to yield a plasmid-free derivative (vii). (Reprinted by permission from Oxford University Press, © 2012).