What is Recombineering?

Recombineering refers to the strategic use of recombination in vivo in order to reach a defined goal. In the case of BioBytes, a method is required to target the final construct to insertion at a specific place on the E. coli chromosome.

To do this successfully, three components must be taken into account:

• There must be a system for targeting the construct to a specific site for insertion

• Activation of the recombination system must be under experimenter control

• It must be possible to select for and verify colonies in which the insertion was successful

Targeting

The BioBytes team has chosen to use a recombination system from bacteriophage lambda. Lambda Red recombinase specifically recombines on the ends of a linear fragment of DNA. If the ends of this fragment are homologous to two separate sites on the E. coli chomosome, the genetic material between these two homologous regions will be exchanged. This will be the basis for our targeting system.

The homologous regions must be a minimum of 50 base pairs in length for recombination to occur at a significant frequency. This can be achieved in different ways:

First 5' extensions corresponding to the homologous sequence can be added to any gene using PCR amplification. This would allow a PCR product to be targeted to a specific site for insertion. Because our constructs will be recircularized and grown (see DNA assembly), this would require us to PCR each plasmid construct seperately in order to add the homologous regions to the ends.

An alternative is to use the genes on either end of our construct as the homologous regions. As an example, we could first locate a region of genes which were deemed inessential through literature review and our Matlab modelling. This region would necessarily be flanked by an essential gene at either end. We would then assemble a plasmid containing these two essential genes. If the insertion is successful, we would be left with a chromosome without this region of inessential genes.

Inducible Recombination System

The Lambda Red Recombinase system consists primarily of three proteins: lambda exonuclease, which processively digests the 5'-ended strand of a dsDNA end; beta protein, which binds to ssDNA and promotes strand annealing; and gamma protein, which binds to the bacterial RecBCD enzyme and inhibits its activities. These three genes are contained on the plasmid pKD46 under an arabinose promoter. For further regulation, this plasmid contains the RepA temperature sensitive origin. Therefore, one can specifically induce recombination of a desired linear piece of DNA, then cure the cell of the pKD46 plasmid at 42C. This system leaves a host cell with a specific chromosal mutation, but is free of the Lambda Red system in order to prevent random, unwanted recombination within the cell.