When short, linear DNA fragments are introduced to enteric bacteria, they are rapidly degraded by exonucleases. However, some phage make inhibitors of the major exonuclease encoded by recBCD. In addition, phage also express gene products that can substitute for the host recombination proteins. In contrast to the host recombination system, the phage recombinases only require short regions of homology (20-50 bp) to promote homologous recombination.
Insights from their studies on the lambda red and P22 erf recombination systems led Murphy and Poteete to develop a system for recombination of short, linear DNA fragments in E. coli. Subsequently, work by two groups, Court + colleagues and Datsenko + Wanner, greatly increased the efficiency of the lambda red recombination system. Both approaches place the lambda red recombinase system under control of a regulated promoter. In the Court approach red is regulated by the temperature sensitive lambda cI857 repressor (induced by growth at 42 C), while in the Datsenko + Wanner approach red is regulated by the araC activator (induced by addition of arabinose). When desired, expression of the recombinase is induced, then the strain is electroporated with a PCR product.
PCR primers are designed so that each end has a 20-50 bp DNA sequence homologous to the chromosomal target sequence, flanking a sequence homologous to a selectable genetic marker (e.g. a kanamycin resistance gene).
Recombination occurs at each end of the PCR product within the region that is homologous to the chromosomal target sequence.
Plating on appropriate plates (in this example, medium with kanamycin) selects for the desired double cross-over recombinants.
Many other genetic tricks have developed which extend the applications of the red recombination system. For example, a two-step protocol can be used to first place a counterselectable marker (e.g. a sacB kan casette or the tetA gene) into a gene via red-mediated recombination, then in a second step the counterselectable marker can be replaced with a "clean" substitution again using red-mediated recombination. It is also possible to construct gene or operon fusions at a precise position in a gene using red-mediated recombination. With a little creativity and a knowledge of genetics, the applications of the red recombination system are (nearly) limitless.
Last modified April 16, 2003