Tag: Calin Plesa

Mapping Sequence-Function Landscapes in the Dihydrofolate Reductase Family Coauthors: Calin Plesa, Samuel Hint

Presenter: Carmen Resnick – Biochemistry

Faculty Mentor(s): Calin Plesa

Session: (In-Person) Poster Presentation

Dihydrofolate reductase (DHFR) is an essential enzyme in the folic acid synthesis pathway and has been the subject of intense study in the past few decades. Despite the wide diversity of homologs, research attention has primarily focused on particular DHFR proteins and as their mutants. In this study we explore DHFR expression through a knockout E. coli strain ER2566 ΔfolAΔthyA. We focus on the ability of DHFR to both rescue metabolic function and tolerate treatment against the antibiotic trimethoprim, which will allow us to understand how antibiotic resistance emerges given many evolutionarily divergent starting points. Changes in the mutational landscape of DHFR allows for varying survival rates in the presence of antibiotic inhibitors. We conduct a broad mutational scan using a library of 5,000 DHFR homologs synthesized using DropSynth gene synthesis. Variant fitness is determined in a multiplex survival assay in the knockout strain which allows supplementation- dependent conditional selection. We aim to collect quantitative fitness data on which mutations impact DHFR activity, both in the presence and absence of inhibitors, to elucidate sequence-function relationships and understand how the fitness landscapes vary as a function of the evolutionary distance between homologs. This data can be applied towards the development of narrow-spectrum and targeted antibiotics and mitigation of resistance through understanding the pathways from which antibiotic resistance arises.

DiversiPhi29—An Orthogonal System for the Continuous Directed Evolution of Genes In Vivo

Presenter: Amanda Kreppel − Biology

Faculty Mentor(s): Nora Kearns, Calin Plesa

Session: (In-Person) Poster Presentation

Directed evolution is a method for protein engineering which allows scientists to impose novel functions on proteins through the random and progressive introduction of mutations to their encoding gene. Traditional directed evolution approaches are inefficient, alternating cycles of manual in vitro mutation and in vivo expression and selection until a desirable advancement in protein function occurs. This limits the throughput and depth at which a protein’s mutational landscape can be explored. By eliminating in vitro mutagenesis and allowing an orthogonal error-prone polymerase to replicate a gene of interest over several generations in E. coli, we are able to push the boundaries of evolution and create large libraries of desirable mutants in vivo. Here we propose DiversiPhi29, which repurposes the replication machinery of bacteriophage ɸ29 to continuously replicate a linear plasmid (pL) carrying a gene of interest in vivo independently of host replication. Once orthogonal replication of pL is established, we will implement a system capable of tuning the mutation rate of the linear construct’s replication by altering the ratio of two ɸ29 DNA Polymerases, one of which contains error-prone mutations. This approach will enable high throughput molecular evolution in the best understood host organism.