Team:Alberta/Project/Recombineering

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University of Alberta - BioBytes










































































































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 for 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 of 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 the ends of a linear fragment of DNA with homologous chromosomal DNA. If the ends of a fragment contain at least 50 bp of homology to two separate sites on the E. coli chromosome, the genetic material between these two homologous regions will be exchanged. This provides the basis for our chromosome assembly 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:

  • 5' extensions corresponding to the homologous sequence can be added to any gene using PCR amplification. However, because our synthetic constructs are rather large and long-extension PCR proves rather finicky, PCRing across our entire linear contructs in order to add homology regions appears rather illogical.
  • An alternative is to use the flanking essential genes on either end of our linear construct as the homologous regions. This provides much greater sequence homology and should provide increased recombination efficiency.

Inducible Recombination System

The Lambda Red Recombinase system consists primarily of three proteins: lambda exonuclease, which progressively 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 chromosomal mutation, but is free of the Lambda Red system in order to prevent future random, unwanted recombination within the cell.

Our Efforts at Recombineering

Attempt I:

Our first attempt at recombineering entailed the insertion of an ampicillin resistance cassette into a gene-less region of the host chromosome. Amp cassette primers were engineered with 50 bp extensions that were homologous to flanking portions of the region of the chromosome to be excised. The linear construct produced by these primers was then electroporated into pre-Arabinose induced pKD46 containing competent cells. Cells were left to incubate at 30C for four hours, then plated on ampicillin containing agarose plates.

Colonies with proper recombination were selected for by PCR verification. PCRing across the excised region was expected to show markedly different fragment sizes between wildtype and ampicillin resistant cells, as the ampicillin cassette was a substantially different size than the region excised.

PCR verification showed no difference in fragment size between wildtype (not ampicillin resistant) and ampicillin resistant cells. This has lead us to believe that non-specific recombination occurred and that the 50 bp of homology used was not great enough for site-specific recombination using our methods.

Attempt II:

In order to decrease the degree of non-specific recombination, we attempted to insert a linear construct with whole-gene homology to the chromosome. To do this, we flanked a chloramphenicol resistance cassette with an essential gene on either side. The region selected for excision contained no known essential genes.

Our 5 byte construct was built using our Biobyte Assembly System and looked like:

  • orn - 75% promoter - amp - 75% promoter - yjeE

The gel purified construct was once again electroporated into pre-Arabinose induced pKD46 containing competent cells. Cells were left to incubate at 30C for four hours, then plated on chloramphenicol containing agarose plates. This time no colonies grew.

This shows that either recombination did not occur, or the cassette was non-functional. We did not have time for further trouble-shooting.

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