Microbial genetics

Phage λ recombination functions (exo, bet, gam) expressed from an inducible plasmid let a linear PCR product with 35–50 bp homology tails replace a chromosomal gene, so a deletion can be made with nothing more than a pair of long primers and an electroporation.

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Lambda Red

How it differs from the standard method

Gam blocks RecBCD so the linear substrate survives; Exo chews back one strand and Beta anneals the single-stranded end to the target.
Short homology on the primer is the whole point, but under about 30 bp efficiency collapses.
The plasmid template of the PCR carries the same resistance gene as the cassette, so DpnI digestion or gel purification is what keeps the plate interpretable.
The helper plasmid is temperature-sensitive for replication and is cured at 37–43 °C once the mutant is confirmed.

In this lesson · about 4 min read

Changing a chromosome, not a plasmid
The three routes, and why they differ
Homology length is a threshold, not a dial
Two crossovers, and the half that go backwards
Key terms
When to use it
Sub-techniques

1Changing a chromosome, not a plasmid

Microbial genetics is the business of altering the chromosome a cell carries, so that the change is stable, single-copy and inherited without selection. The tool in every version of it is homologous recombination: a piece of DNA that matches the target locus at both ends is brought into the cell, the cell's own machinery (or a borrowed phage system) lines the matching sequences up, and the stretch between them is swapped for whatever you put there.

2The three routes, and why they differ

Lambda Red

Borrowed recombinases

Gam, Beta and Exo from phage λ, expressed from an inducible plasmid, recombine a linear PCR product with as little as 35–50 bp of flanking homology: short enough to carry on a primer.

Yeast

The cell's own machinery

S. cerevisiae recombines transformed DNA at its homologue efficiently. 45 bp arms work; 300–500 bp arms work roughly an order of magnitude better and cut the off-target rate.

Conjugation

Delivery by mating

For organisms you cannot electroporate, a suicide plasmid is mated in from an E. coli donor, integrates by a single crossover, and is then forced out again by counter-selection.

Unmarking

Getting the cassette back out

FLP recombinase acting on FRT sites either side of the cassette leaves a short scar, so the marker is free for the next round and no promoter is left reading into the neighbour.

3Homology length is a threshold, not a dial

How much flanking homology you need depends entirely on which machinery is doing the work. The λ Red annealase works on single-stranded ends and tolerates very short matches, which is why recombineering can be done with nothing but a pair of long primers; below about 30 bp, though, efficiency does not decline gently, it collapses.

4Two crossovers, and the half that go backwards

Conjugation-based allelic exchange is a two-step selection and it is worth following through. The suicide plasmid cannot replicate in the recipient, so the only cells that keep its antibiotic marker are those where a single crossover has integrated the whole plasmid into the chromosome.

Merodiploid: A cell carrying two copies of a locus, usually wild-type and mutant flanking an integrated plasmid. An intermediate, not a mutant.
Suicide plasmid: A vector with no origin that works in the recipient, so it is lost unless it integrates. The basis of selecting a single crossover.
Counter-selection: A condition that kills cells which still carry a marker (sacB in sucrose, URA3 in 5-fluoroorotic acid, rpsL, thyA), used to force the plasmid out.
Merged background: Colonies that are resistant for reasons other than the recombination you intended: template plasmid, off-target insertion, spontaneous resistance.

Key terms

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Recombineering

Recombination-mediated genetic engineering: using phage recombinases (λ Red, or RecET) so that linear DNA with short homology arms recombines with the chromosome.

FRT scar

The 80-odd bases left behind when FLP excises a cassette between two FRT sites. Unmarked, but not seamless.

oriT

The origin of transfer on a conjugative or mobilisable plasmid: the sequence a donor's transfer machinery nicks and pumps into a recipient.

Dominant marker

A resistance gene that works in any strain (kanMX/G418, nat, hph), as opposed to an auxotrophic marker that complements a mutation the strain already carries.

Junction PCR

A confirmation PCR with one primer outside the homology arm and one inside the cassette, so it only amplifies if the cassette sits at the target locus.

Merodiploid resolution

The second crossover that excises an integrated plasmid, leaving either the new allele or the original one behind.

When to use it

You need a stable, single-copy, inherited change: a deletion, a point mutation, a tag or a promoter swap in the chromosome rather than on a plasmid.
You want an unmarked mutant for a phenotype study, where a plasmid's copy number or an antibiotic in the medium would confound the result.
The organism cannot be transformed by electroporation or chemical competence, and DNA has to be delivered by mating instead.
You are building a strain series where each round must re-use the same marker, so the cassette has to come out again.

Compared with alternatives

Bacterial transformation →

Use plain transformation when the construct can live on a plasmid: far higher efficiency, no recombination step, and nothing to confirm beyond the plasmid itself.

CRISPR-Cas9 editing →

Prefer a nuclease-based edit when you need a scarless point mutation at high frequency, or when no selectable marker can be left behind; the cut makes recombination with a short template far more likely.

Other editing & transgenesis →

Transposon and integrase systems are the better choice when you want insertion anywhere, or at a defined attachment site, rather than replacement of a specific locus.

Plating & streaking →

Every one of these constructions ends on a plate, and the single-colony purification that makes a strain clonal is plating technique, not genetics.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

Gam blocks RecBCD so the linear substrate survives; Exo chews back one strand and Beta anneals the single-stranded end to the target.
Short homology on the primer is the whole point, but under about 30 bp efficiency collapses.
The plasmid template of the PCR carries the same resistance gene as the cassette, so DpnI digestion or gel purification is what keeps the plate interpretable.
The helper plasmid is temperature-sensitive for replication and is cured at 37–43 °C once the mutant is confirmed.