Recombineering: Genetic Engineering in Escherichia coli Using Homologous Recombination

Abstract

The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using either PCR products or synthetic double-stranded DNA (dsDNA) or single-stranded DNA as substrates. Multiple linear dsDNA molecules can be assembled into an intact plasmid. The technology of recombineering is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to the location of restriction sites and can also be used in combination with CRISPR/Cas targeting systems. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: Making electrocompetent cells and transforming with linear DNA Support Protocol 1: Selection/counter-selections for genome engineering Support Protocol 2: Creating and screening for oligo recombinants by PCR Support Protocol 3: Other methods of screening for unselected recombinants Support Protocol 4: Curing recombineering plasmids containing a temperature-sensitive replication function Support Protocol 5: Removal of the prophage by recombineering Alternate Protocol 1: Using CRISPR/Cas9 as a counter-selection following recombineering Alternate Protocol 2: Assembly of linear dsDNA fragments into functional plasmids Alternate Protocol 3: Retrieval of alleles onto a plasmid by gap repair Alternate Protocol 4: Modifying multicopy plasmids with recombineering Support Protocol 6: Screening for unselected plasmid recombinants Alternate Protocol 5: Recombineering with an intact λ prophage Alternate Protocol 6: Targeting an infecting λ phage with the defective prophage strains.

Keywords: Escherichia coli; Rac prophage RecET; bacteriophage λ; homologous recombination; recombineering; λ Red system.

Figure 1.. A general flow chart for genomic and BAC recombineering.

General steps are shown for making point mutations or larger DNA insertions on the E. coli genome or a BAC, as well as two-step method allowing the insertion of un-selectable DNA into the genome. (Several two-step options are provided in Support Protocol 1 and Figure 7). Large deletions can be made with oligos, but the frequency of recovery is lower than that of point mutations and a selection is required. Multicopy plasmids can also be targeted with either ssDNA or ssDNA substrates, but complications can arise (see Alternative Protocol 4 and Support Protocol 6 for details).

Figure 2.. A general flow chart for plasmid recombineering.

General steps are shown for assembly of dsDNA linear DNA into intact plasmids and for rescue of genomic DNA onto a plasmid backbone i.e. cloning by gene retrieval. Note that when targeting recombineering to plasmids, it is important to use bacterial strains expressing the recombination system from the defective prophage rather than from a plasmid.

Figure 3.. Molecular Mechanism of Recombineering.

Red recombination occurs at the bacterial or plasmid DNA replication fork, at single-strand gaps arising during DNA replication. A. A ssDNA oligo with a single mismatch (i.e. for making a point mutation) annealed at a complementary region of the target. Such a complex is generally subject to E.coli Methyl-directed Mismatch Repair (MMR) (indicated by the yellow Pacman); only C-C mispairs are not repaired by MMR. MMR repair lowers efficiency of recombineering ~100-fold. B. In contrast, an oligo with multiple mismatches can still anneal to the complementary target, but the larger distortion in the DNA/oligo complex prevents MMR from occurring and results in higher recombination efficiencies. C. A schematic of the DNA replication fork with leading and lagging strands indicated. A ssDNA oligonucleotide, coated by the λ Beta protein, is shown in the process of being incorporated into the discontinuously replicated lagging strand at a single strand gap. D. Illustration of leading vs lagging oligo choice. To determine which strand is lagging and which is leading, first identify both the origin of DNA replication and the terminus for your replicon (the terminus will be approximately opposite from the origin). The E. coli origin is bi-directional, and the terminus has been defined (Blattner et al., 1997). To the right of the origin, and between the origin and the terminus, Ellis et al (2001) found that 5’ to 3’ oligos pointing counterclockwise towards the ori have a higher recombination frequency than those pointing clockwise, away from the ori. These 5’ to 3’ oligos that point counterclockwise towards the origin correspond to the lagging strand, and are thus lagging strand oligos, which recombine with a higher efficiency. Those that point clockwise towards the terminus correspond to the leading strand and are thus leading strand oligos, which recombine with a lower efficiency. To the left of the origin, the situation is reversed: oligos between the origin and the terminus that point clockwise towards the ori are the high efficiency lagging strand oligos, and those pointing counterclockwise are lower efficiency leading strand oligos.

Figure 4.. Two types of plasmid recombineering reactions.

A. Schematic illustrating linear DNA assembly. Linear pieces of dsDNA with 30–50 nt terminal homologies, the direction of which is indicated by the arrows at the end of the DNA fragments, are introduced by electroporation into cells expressing the RecET system. RecET recombines the individual pieces into an intact plasmid accurately and efficiently. B. Retrieval onto a plasmid by gap repair. A linear plasmid backbone containing an antibiotic resistance gene and an ori with flanking homology to the target at the ends (the direction is indicated by cyan and purple arrows) is generated by PCR. The plasmid is introduced by electroporation into cells expressing the Red functions, which catalyze recombination of the vector with the target site, resulting in incorporation of the region of interest (indicated by the yellow arrow) onto the plasmid. Note that targeting of intact plasmids with recombineering (Alternate Protocol 4) is not shown here.

Figure 5.. PCR generation of an antibiotic cassette for gene targeting.

Two ~ 70 nt primers with 5′ homology to the target, shown in cyan and purple, are used for PCR amplification of the antibiotic cassette, ideally using the technique of colony PCR and a bacterial strain such as T-SACK as a template. The PCR product is purified and introduced by electroporation into cells induced for the Red recombineering functions. The Red functions catalyze the insertion of the cassette at the target site, which may be on the bacterial chromosome or on a plasmid.

Figure 6.. Expression of the Red and RecET systems from the defective prophage and pSIM plasmids.

A, B. In most of the recombineering strains available from the Court laboratory, the three λ Red genes, exo, bet, and gam, are expressed from the powerful native phage λ P_L promoter. C, D. This same regulatory system is used to express gam, recE and recT in strains NC553 and LT1795. At low temperatures (30°C to 32°C), the temperature-sensitive λ CI857 repressor binds cooperatively to operator sites adjacent to the promoter and the P_L promoter is tightly repressed. Raising the temperature to 42°C results in rapid inactivation of the repressor protein and high-level expression of the recombination functions. Lowering the temperature after a 15-min induction allows CI857 to renature with consequent restoration of tight repression. A second set of operator sites is present at the phage λ P_R promoter, located beyond the cI repressor gene. When both sets of operators are present, as they are in the Court laboratory recombineering constructs, protein-protein interactions between CI repressor molecules bound to the operator sites act to loop the DNA between the two sets of operators (Dodd et al. 2005), and this looping provides an additional layer of P_L repression (Lewis et al, 2016).

Figure 7. A flow chart for two-step selection/counter-selections.

Three procedures are provided, two procedures using the sacB counter-selectable gene linked to either of two antibiotic resistance genes, cat or tetA, and one procedure using galK, that is designed specifically for BAC modification.