Designing technologies for engineering cells: Targeted, continuous evolution and organelle compartmentalization
John Dueber, University of California, Berkeley
12:00pm - 1:00pm
Location: 106, Stanley Hall
My research group aims to reprogram living cells for engineering applications. In this talk, I will discuss two main areas of focus. First, in a collaboration with David Schaffer, we have developed a technology, EvolvR, to harness the cell’s ability to evolve in a more focused, accelerated manner. EvolvR consists of a nicking Cas9 (nCas9) tethered to a nick-translating DNA polymerase (DNAP). nCas9 provides guide RNA-mediated targeting to desired plasmid or genomic loci while various DNAPs can be used to tune the fidelity and processivity for incorporating mutations. EvolvR promises the ability, provided a constant selective pressure, to continuously diversify defined loci to allow exploration of evolutionary paths requiring sequential mutation events. Additionally, EvolvR should be portable from our original test host, E. coli, to organisms where transformation and homologous recombination efficiencies are low. Second, we are aiming to gain spatial control over cellular processes by engineering a synthetic organelle. We are repurposing the peroxisome in yeast as this organelle is not required for viability, is capable of importing high quantities of folded protein, and has natural precedent of having flexibility in what cellular processes can be compartmentalized. We have engineered improved soluble protein targeting and an orthogonal mechanism for modularly and functionally trafficking plasma and vacuole membrane proteins to the peroxisome membrane. For both of these research focus areas, I will briefly outline ideas where we envision taking these projects.