Keasling Lab

Background

One of the challenges of using recombination events for the in vivo construction of metabolic pathways is the reusability of recognition sites for further insertion and excision events.  While this can allow for multiple desired insertions into the same site, excisions that remove multiple genes in a single block are possible.  To combat this, we are studying a number of site specific recombinases and recognition sites that can be biased to favor insertion over excision, as well as identifying enzymes that can function simultaneously in vivo in order to increase the number of independently targetable insertion sites. This will allow us to break a pathway into separate sections (i.e. backbone construction, cyclization, hydroxylation, etc), improving the likelihood of generating a functional pathway while enabling an increase in the number of gene candidates per function.

Current Focus

We have identified a number of enzymes that can perform cassette manipulation simultaneously with low levels of cross-reactivity.  We are currently scaling up our search methodology to identify additional enzymes and recognitions sites and to have a better understanding of what products are likely when cross reactivity does occur.  Understanding these interactions will allow us to prioritize our multi-enzyme systems to lower the failure rate and may allow for the design of more complex recombination-based devices.

People

  • Prof. Jay Keasling
  • Dr. Josh Gilmore

Related publications

Links