P lab

Engineering Improved DNA Ligases

DNA ligase enzymes catalyze the formation of new phosphodiester bonds in DNA; that is, they join one linear DNA molecule to another. Nearly all molecular biologists use a particular enzyme, T4 DNA ligase, to create recombinant DNA molecules (i.e. to ‘clone’ DNA fragments in vitro). Next-generation DNA sequencing technologies also use DNA ligase in their library construction and/or sequencing steps. This makes T4 DNA ligase one of the most widely-used enzymes in the world, with an estimated global market of > US$20 million per annum. However, DNA ligase did not evolve to carry out our in vitro reactions, and ligation is almost always the rate-limiting step in experimental protocols.

We are attempting to engineer improved versions of T4 DNA ligase, by fusing it to a variety of DNA-binding proteins. Several of the fusion proteins out-perform the unmodified DNA ligase, particularly in blunt-ended ligation reactions. A slightly cheesy account of the research can be downloaded here (PDF, 364 kB). We are also identifying new DNA ligases, through a combination of bio-prospecting and ancestral sequence reconstruction. This work is being funded by an MBIE Smart Ideas grant.

     
 
Ligase assay
 
 
Gel‐based activity assay. DNA ligase activity is measured as the disappearance of the 717 bp substrate band, and the appearance of the ligated product (1,434 bp). Lanes 1‐10 are reactions containing modified ligases. The lane labelled M is a molecular weight ladder. T4 is the reaction containing unmodified T4 DNA ligase; the negative control (“‐ve”) contained no ligase in the reaction.