How to become a "recombineer", today!
What is “recombineering”?
Recombineering is a molecular biology technique based on the phenomenon of homologous recombination (nucleotide sequences are exchanged between similar or identical DNA molecules, most often used by cells to repair double stranded DNA breaks, as well as to introduce variation during gamete production).
The technique is used in place of the more common restriction enzyme digestions and ligations to combine DNA sequences in a specified order.
It has been used to study bacterial genetics and construct recombinant DNA in order to generate transgenic and knockout mice.
What is “lambda red” recombineering?
The name comes from the lambda phage (virus that infects and replicates inside E. coli) proteins that mediate homologous recombination in E. coli. This is now the most commonly used in vivo (meaning that the whole organism is used in the experiment) recombineering technique, and was demonstrated by Francis Stewart, in the late 90’s.
In lambda red recombineering, linear DNA fragments are transformed into E. coli cells, and then integrated into the bacterial chromosome via homologous recombination.
Let’s back up and discuss homologous recombination, in the context of double-stranded DNA break repair in bacteria, which have circular DNA.
- When a double-stranded DNA break occurs, resulting in a linear piece of DNA, the enzyme RecBCD binds at the breakage point and trims the ends to generate overhangs (see figure below). The overhangs are recognized and covered by the RecA protein, which, when it finds a similar DNA sequence, mediates something called strand invasion.
- Strand invasion occurs when one of the overhanging strands of DNA causes a strand of the recipient DNA complex to be displaced. The resulting “X” is called a Holliday junction. The gaps are filled in by DNA synthesis.
- The next step is branch migration, the process by which the Holliday junction migrates further down the DNA strand, catalyzed by two proteins, called RuvA and Ruv B.
- The Holliday junction is resolved, or released, when RucC cleaves the crossed strands, which are then joined by DNA ligase.
In the context of lambda red recombineering, I mentioned that linear DNA fragments are integrated into the bacterial chromosome by homologous recombination.
But, doesn’t RecBCD degrade linear double-stranded DNA?
Right, so E. coli is not easily transformed with linear DNA fragments. Enter, the lambda bacteriophage. This virus has its own recombination system, which mediates the integration of its linear DNA into the E. coli chromosome during its lifecycle.
The phage’s recombination system is made up of three proteins, one of which binds RecBCD and inhibits its activity. The other two proteins function to generate overhangs, facilitate strand invasion, and annealing between complementary strands.
What can we do with this?
Remember, we’re talking about homologous recombination, so there has to be a region of similar DNA in the mix; we can replace a gene with an antibiotic resistance gene, as long as we generate a PCR product that has flanking regions of homology on either side of the gene.
Or, we can fuse a fluorescent tag to our gene of interest. Here I’ve used GFP flanked by 35 nucleotides of homology to our gene, and 35 nucleotides of homology to the chromosome.
How does this work?
There are two methods to exploiting this system:
- Expression of lambda red protein, or
- Modification of a bacterial artificial chromosome (BAC), which is as it sounds – an artificial DNA construct – using galk positive and negative selection.
I will only explain the second method here.
The galk gene encodes galactokinase, which is critical for galactose metabolism. If galk is active, the bacteria can grow on galactose media. Thus, this can be used for positive selection, as you’ll see in a moment.
However, galactokinase can also phosphorylate 2-deoxy-galactose (DOG) into DOG-P, which is toxic for cells. Thus, cells that do not have galk can grow on DOG media, resulting in negative selection.
The cells used in this method express lambda phage proteins, but lack the galk gene, so that they cannot grow on galactose media.
For example, to introduce a mutation in your gene of interest:
What are the advantages of this system?
- Eliminates the need for precisely positioned restriction sites.
- Can manipulate large constructs of DNA.
- Forgoes multiple cloning stages, and thus can be done in a relatively short time-frame.
- Highly efficient.
Congratulations! You are now equipped with an understanding of the technique, and with this, you can become a “recombineer”!