Friday, April 4, 2008

Recombineering: A Practical Application of Phage Biology

ResearchBlogging.orgThe most obvious use of phages is of course direct phage therapy. Although exciting, there are many other uses of phage that are just as revolutionary, but tend to slip by. In my last article, I hinted at (Short for homology-dependent, recombination-mediated, genetic engineering) This is a method whereby phage proteins are used to catalyze homologous recombination. The ramifications are huge. Mutations can be made; whole genes and operons can be knocked out with relative ease, and much more. In fact, some may say that this recently described process revolutionized genetics.

Since this system was so successful in E. coli, the Hatfull Lab at the University of Pittsburgh set out to develop this system in the . Before this, genetic manipulation (especially gene knockouts) was highly difficult. M. tuberculosis is notoriously recalcitrant to genetic manipulation. This is due to a variety of factors including: its horribly slow growth rate, high pathogenicity, and its relatively high levels of illegitimate recombination compared to homologous recombination.

This paper, by vanKessel and Hatfull was published relatively recently (Jan 2007) and describes what is a great advance in mycobacterial genetics.

The first step was to identify potential recombination proteins encoded by phages. These proteins are presumed to be necessary for proper facilitation of lysogenic integration and DNA replication. Looking at the functions of the “Red” recombination system, (used for recombineering) one can find functional homologs in other systems. The Red system uses three proteins: exo, beta, and gam. Exo acts as a 5’-3’ dsDNA dependant exonuclease, beta acts to bind ssDNA and promote annealing, and gam functions to inactivate an alternative host recombination pathway (the RecBCD system).

Using this as the basis for comparison, one first comes across the Rac of E. coli. This prophage encodes two proteins called RecE and RecT that function identically to the exo and beta genes respectivly in the Red system, without significant sequence similarity. One then can look at the published mycobacteriophage genomes (30 at the time of publication, soon to be 50) and search for functional analogs of either the Red or Rac system. The authors found that in one phage of the 30, recET homologs were found. This phage, Che9c, contains two genes that are each around 30% identical to the Rac prophage RecE and RecT (at Che9c gp60 and 61).

Biochemical analysis showed that RecE and RecT function nearly identically to the Lambda phage exo and beta.

The authors placed Che9c gp60 and 61 onto a plasmid under an acetamide promoter (high expression of these genes under a constitutive promoter was determined to be toxic) and transformed into M. smegmatis (a fast growing, non-pathogenic relative to M. tuberculosis)

These cells could then be transformed with a dsDNA leuD knockout substrate containing 1,000bp homology on each side (500bp flanking sequence and 500bp leuD sequence) of a Hyg resistance cassette. Upon induction of the RecET homologs, allelic exchange was shown to occur at a measureable frequence (8x10^-4 recombinants per ug / cell competancy) by detecting the presence of leucine auxotrophs with hygromycin resistance. This event was shown to require both Che9c gp60 and gp61, indicating that both RecE and RecT functions were needed.

This method was shown to work not only in M. smegmatis, but also in M. tuberculosis and at different genetic loci.

Other work showed that 1,000bp homology was not necessarily needed and that substrates could have as little as 50bp (the current standard is now 500bp homology) flanking sequence to the knock out marker.

This method will certainly revolutionize the ability to genetically manipulate the Mycobacteria. The possibilities are endless. Not only can one create knockouts, but also insert point mutations, amber mutants, add frameshifts, and add His-Tags. This is not to mention that all these methods can be applied not only to the bacterial genome, but also phage.

This is a wonderful example of phage-based technology with a practical and immensely helpful function.
van Kessel, J.C., Hatfull, G.F. (2007). Recombineering in Mycobacterium tuberculosis. Nature Methods, 4(2), 147-152. DOI: 10.1038/nmeth996

Book Recommendations

For more information regarding how we work woth the Mycobacteria, this "handbook" describes all the major protocols.
Mycobacteria Protocols (Methods in Molecular Biology) (Methods in Molecular Biology)

As always, I recommend this book for anyone interested in a well described overview of mycobacterial genetics.
Molecular Genetics Mycobacteria

Finally, this is one of my favorite books on bacteriophages. It describes all the major topics in phage biology and their applications.
The Bacteriophages

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