Over the years, scientists learned to exploit many aspects of genetic engineering using bacteriophages, in addition to transduction.

One such method is Lambda Red Mediated Recombineering, which is a method based on homologous recombination. This system uses proteins from the lambda bacteriophage, which facilitate homologous recombination between a foreign DNA fragment of interest and the host bacterial genome.

In this article:

Significance of the “Red” terminology

Basic Lambda Red process

Lambda Red workflow in detail

Synthesis and electroporation of desired fragment

Homologous recombination event mediated by lambda Red proteins

Expression of the selectable marker in the recombinant host bacterial cells

Selection of recombinants

Colony PCR to confirm recombinants

Confirming recombinants based on primer approach - what to look for

How are Red proteins expressed in the recipient host bacteria?

Red expression method 1: Plasmid

Red expression method 2: From the host chromosome

Red expression method 3: Mini-lambda

Red expression method 4: Using the lambda phage

References

Homologous recombination is a process of genetic exchange. Here, recombination of genetic sequences occurs between two different strands of nucleic acids having some degree of similarity (that is, the two sequences are homologous).

Illustration of homologous recombination taking place between a linear PCR fragment and a chromosomal DNA.


Figure 1. Illustration of homologous recombination taking place between a linear PCR fragment and a chromosomal DNA.

This process of genetic engineering based on homologous recombination, is known as “recombineering” (recombination-mediated genetic engineering). Recombineering has been discovered and standardized using the model bacterium and bacteriophage pair E. coli and lambda phage.

Significance of the “Red” terminology

For the term “Lambda Red Recombineering,” we now understand the significance of “lambda” and “recombineering,” but what is “Red”? Let us answer that here.

Almost 50 years ago, scientists figured out they could use certain proteins of the bacteriophage lambda to achieve homologous recombination, even in a mutant E. coli strain that lacked its own homologous recombination machinery.

This new DNA recombination system, composed of lambda phage proteins, is known as, “Red” as an abbreviation for “recombination defective.” The “Red” term highlights the uniqueness of this system from E. coli’s own recombination machinery.

Thus, taken together, since this system mechanistically involves recombineering using lambda’s Red multi-protein machinery for genetic manipulation in bacteria, it is termed as “Lambda Red Recombineering.”

The usual workflow involves creating selectable mutations in an E. coli strain using lambda Red proteins. Then, using that mutated strain, it is used as the donor to transduce using the P1vir selectable mutation into a desired bacterial strain, called the recipient.

Basic Lambda Red process

The lambda Red recombineering procedure, as typically performed in regular laboratory practice, is depicted in Figure 2. Here is a quick glance of the main four steps in this process:

  • DNA fragments intended to be introduced into the host cells’ chromosomes are synthesized using PCR. These synthesized fragments are then introduced into the host cell. Generally, the introduced DNA fragments have a selectable marker such as an antibiotic resistance cassette.
  • The fragments recombine with the corresponding homologous region of the host chromosome using lambda Red proteins.
  • Researchers account for phenotypic lag for expression of the selectable marker, which is within the new fragment incorporated in the host genome. After that, the host bacterial culture is plated on selective media to select recombinants.
  • Finally, colony PCR is used to confirm recombinants.

Illustration of lambda Red recombineering workflow

Figure 2. Illustration of lambda Red recombineering workflow. Note that recombinant cells (green) are the only ones that will survive being plate on selective media. Nonrecombinant cells (orange) will not produce colonies.


Lambda Red workflow in detail

There are five basic steps to the lambda Red workflow:

  • fragment synthesis and electroporation
  • homologous recombination
  • expression of the selectable marker
  • recombinant selection
  • recombinant confirmation

We’ll take a look, in detail, at what is involved with each of these steps.

Synthesis and electroporation of the desired fragment

A linear PCR fragment is electroporated into the host cell. This linear DNA fragment has two important features. First, the 5’ and 3’ ends bear homology to the sequences flanking the fragment of interest (FoI), which is usually a gene, in the host cell’s chromosome.

Second, the central part of the PCR fragment comprises the sequence that the fragment of interest in the host chromosome is intended to be swapped with.

For a gene deletion, this sequence is commonly an antibiotic resistance cassette. If, instead, a point mutation is wanted in the fragment of interest, this sequence would be the fragment of interest but with that mutation.


Tips and considerations when doing synthesis and electroporation

  • >40bp of homology is required at both ends of the PCR fragment with upstream and downstream regions of the host chromosome that flank the fragment of interest.
  • For PCR products with short homologies, increased levels of Red protein functions are required for efficient recombineering.
  • For the PCR reaction, intact plasmids (the whole plasmid vector that can replicate once introduced into the host cell) are not recommended to be used as templates, especially for amplifying antibiotic resistance cassettes. Such plasmids might get into the recipient host during electroporation at high transformation efficiency and produce false positives during selection. Chromosomally located antibiotic resistance genes may be used as PCR templates.
  • In case a plasmid indeed must be used as a template for the PCR, it is recommended you treat the PCR product with an enzyme such as DpnI, which is an enzyme that digests only methylated DNA such as a plasmid, prior to using it for electroporation.
  • For electroporation, 1-3µl of DNA (~ 50 – 300 ng)is recommended per 50 µl of cells.

Homologous recombination event mediated by lambda Red proteins


Tips and considerations when doing synthesis and electroporation ●>40bp of homology is required at both ends of the PCR fragment with upstream and downstream regions of the host chromosome that flank the fragment of interest. ●	For PCR products with short homologies, increased levels of Red protein functions are required for efficient recombineering. ●	For the PCR reaction, intact plasmids (the whole plasmid vector that can replicate once introduced into the host cell) are not recommended to be used as templates, especially for amplifying antibiotic resistance cassettes. Such plasmids might get into the recipient host during electroporation at high transformation efficiency and produce false positives during selection. Chromosomally located antibiotic resistance genes may be used as PCR templates. ●	In case a plasmid indeed must be used as a template for the PCR, it is recommended you treat the PCR product with an enzyme such as DpnI, which is an enzyme that digests only methylated DNA such as a plasmid, prior to using it for electroporation. ●	For electroporation, 1-3µl of DNA  (~ 50 – 300 ng)is recommended per 50 µl of cells.   Homologous recombination event mediated by lambda Red proteins

Figure 3. Representation of the lambda Red proteins RecBCD, Gam and SbcCD. The text below discusses each protein’s function in greater detail, as well as highlights relevant elements of this image.

The Red proteins facilitate recombination between the PCR fragment and fragment of interest in the host chromosome. Below, we will talk about important proteins within the lambda Red system.


Gam protein

Lambda's Gam proteins prevent host cell nucleases (enzymes that digest nucleic acids) RecBCD and SbcCD from degrading the newly introduced PCR fragment. Gam directly binds to the host nuclease machinery RecBCD and blocks it from binding (and subsequently digesting) the electroporated double-stranded PCR fragment.


Exo protein

Now, the Exo protein of lambda plays its role as an exonuclease enzyme. It digests the PCR fragment from the 5’ ends.

The enzyme is composed as a trimer of three Exo polypeptides whose central region has a funnel shaped architecture. The double-stranded PCR fragment enters this “funnel” through its wider end. The two DNA strands are separated, and the 5’-ended strand is digested. The 3’-end now becomes single-stranded and comes out of the funnel through the narrow end.

This ultimately results in the PCR fragment becoming a partially double-stranded DNA with 3’-single-stranded overhangs.

Here's a schematic representation of the role of Exo protein.


Beta proteins

Next lambda’s beta protein binds to the 3’-single-stranded DNA and promotes annealing to the corresponding complementary single-strand in the host’s chromosome.

Now it might start to make sense why we designed the PCR product to bear 5’ and 3’ homology to DNA regions flanking the fragment of interest in the host chromosome.

The beta protein promotes and facilitates strand exchange and recombination between the PCR fragment and the target sequence in the host chromosome.


Homologous recombination considerations and tips:

For the fragment of interest, even single-stranded DNA can be used. When using single-stranded DNA from the Red proteins, only beta is necessary for recombineering.

Expression of the selectable marker in the recombinant host bacterial cells

After the recombination takes place, the recipient host is cultured in rich media for about an hour to account for phenotypic lag in expression of the selectable marker. The selectable marker is usually an antibiotic resistance cassette associated with the recombination.

In case you wanted a better understanding of phenotypic lag and its importance while culturing, we have a section all about it in our bacterial transformation article.


Selection of recombinants

Next, the culture is plated on selective media for positive selection of recombinants. Host cells that have indeed undergone the recombination are able to survive the selection pressure, while wild-type host bacteria, which have not undergone recombination for any reason, die out.


The above image is a snippet of figure 2. Here, it depicts this concept of survivability on media with a selection antibiotic. The green cell depicted in the test tube of this image has undergone recombination, and therefore will produce colonies when plated. However, the orange cell represents cells where recombination did not occur for one reason or another. When plated, these orange cells will not produce colonies.


Colony PCR to confirm recombinants

The colonies are streaked one-two times for isolation and confirmed by colony PCR.

For more information about colony PCR, we have some helpful information in this article. It may help to briefly look at this article for context, especially about the primer sets involved and how to choose the primer sets.

In short, there are a few different options for primers, and they differ in where they bind. Each option offers some advantages and disadvantages.

In Figure 4 below, we show examples of what you would observe when confirming recombinants versus the wild type. Each of these examples depend on the primer approach used.


Figure 4. Confirmation of recombinants by colony PCR.


Figure 4. Confirmation of recombinants by colony PCR.

Confirming recombinants based on primer approach - what to look for

When primers 1,2 are used in the PCR (lanes 1-2 from left), they bind just upstream and downstream of the chromosomal locus of interest. Antibiotic resistance cassettes are typically large fragments. Therefore, with primers 1,2, the recombinant colony produces a higher band compared to that produced using the wild type as a template.

Primer 3 is specific for the antibiotic resistance cassette, so PCR of the recombinant colony with primers 1,3 produces a band on the gel (lane 3) while in the case of the wild type, only a primer dimer (fuzzy very low molecular weight band) is produced (lane 4).

Similarly, primer 4 is specific for the antibiotic resistance cassette. Hence, PCR of the recombinant colony with primers 2,4 produces a band on the gel (lane 5) while in the case of wild type, only a primer dimer (fuzzy, very low molecular weight band) is produced (lane 6).

The last lane represents the DNA ladder

How are Red proteins expressed in the recipient host bacteria?

The Red enzymes used in the lambda Red process can be expressed using any one of these methods:

Red expression method 1: Plasmid

Expressing Red proteins encoded by a plasmid introduced into the recipient host cell is the most popular approach.

It is very important to tightly control expression of Red proteins from this plasmid to prevent other unwanted widespread recombinations within the host chromosome. That is, other than the intended gene recombination between the PCR fragment and chromosomal fragment of interest.

To tightly control their expression, the Red proteins are expressed using conditional promoters such as Plac, PBAD etc.

Further, such plasmids often have a heat-sensitive origin of replication. Once the recombineering is completed, the recipient is grown at a higher temperature, usually 42 0C.

The higher temperature results in the elimination of this plasmid from the host cell due to the heat-sensitive origin of replication in the plasmid. This is another helpful feature to prevent spontaneous aberrant mutagenesis that may be caused by the Red enzymes encoded by the plasmid.

Red expression method 2: From the host chromosome

Red proteins can be encoded by the host’s chromosome, often from a defective prophage.

The promoters used are such that the Red proteins are expressed only when the host cell is grown at 42 0 C.

Once recombineering is completed, the Red genes from the host’s chromosome may be eliminated to prevent other mutations.

However, this tends to be a complicated process. To avoid the trouble of eliminating the Red protein coding genes from the host’s chromosome, researchers use P1vir transduction to transduce the selectable mutation achieved by recombineering to another strain, which does not have Red protein expression.


Red expression method 3: Mini-lambda

The mini-lambda expression system is a hybrid between a plasmid and a defective prophage.

Mini-lambda is a non-replicating circular piece of phage DNA encoding the Red proteins. Upon introducing it to the host cell, it integrates into the host cell’s chromosome.

Expression of Red machinery is contingent upon growing the host strain at 42 0C.


Red expression method 4: Using the lambda phage

The lambda phage, with a selectable antibiotic resistance marker, can be used to express Red proteins.

Once introduced into the host, the phage adopts the lysogenic cycle and integrates into the chromosome as a prophage.

The selectable mutation thus engineered in the host bacterial strain is subsequently transduced into a second strain (most commonly the bacterial strain under experimentation) using P1vir transduction.


References

  • Caldwell B.J and Bell C.E (2019). Structure and mechanism of the Red recombination system of bacteriophage λ. Progress in Biophysics and molecular biology.147(33-46). https://doi.org/10.1016/j.pbiomolbio.2019.03.005
  • Murphy K.C (2016). λ recombination and recombineering. EcoSal Plus. 7(1). doi:10.1128/ecosalplus.ESP-0011-2015.
  • Sharan S.K et al. (2009). Recombineering: A homologous recombination-based method of genetic engineering. Nat Protoc. 4(2). doi: 10.1038/nprot.2008.227