Fig. 2 |. Optimizing the ARF in bacteria.

There are several factors that help improve the allelic recombination frequency (ARF), which are applicable across most bacterial species. a | Oligodeoxynucleotides (oligos) are designed containing phosphorothioate (PT) bonds, shown as asterisks. b | A population of bacteria are transformed with oligos through electroporation. The percentage of cells successfully transformed presents an upper bound for the ARF. Once inside the host cell, all free single-stranded DNA (ssDNA) is bound by bacterial single-stranded DNA-binding protein (SSB). c | Host nucleases degrade ssDNA in most bacteria, so protection with PT bonds is important. Two PT bonds at the 5′ end of the oligo usually provide adequate protection. d | Optimization of protein production can improve the ARF significantly. Some factors to consider are codon optimization, promoter strength and ribosome binding site strength. e | Orders of magnitude improvement can be gained by expressing a host-optimized single-stranded DNA-annealing protein (SSAP). A specific interaction with a host SSB determines SSAP compatibility. f | After a modification has been made to the genomic DNA, a mismatched base pair will be present. There are several possible strategies to prevent error correction by endogenous mismatch repair (MMR) machinery. Transient expression of a dominant-negative MutL (MutL-DN) causes chain termination and failure to recruit MutH. This optimal route is depicted here, but alternatives are available including MutH or MutS disruption, and the modification of multiple (4+) consecutive bases, which would then not be efficiently recognized by MutS. g | Serial enrichment for efficient recombineering (SEER) is a method to identify a host-optimized SSAP. A diverse library of SSAP variants is encoded on a plasmid, transformed into the target bacterial host, enriched through a series of antibiotic selections and deep sequenced to identify the most successful SSAP variants. Oligos used in the enrichment steps each contain an allelic modification to a host gene that confers resistance to a common antibiotic. Ab R, antibiotic resistance gene; NGS, next-generation sequencing; Ori, origin of replication.