Mapping the Sequence-Function Landscape for Antibiotic Resistance in the DHFR Family
| dc.contributor.advisor | Plesa, Calin | |
| dc.contributor.advisor | McWhorter, Brian | |
| dc.contributor.author | Resnick, Carmen | |
| dc.date.accessioned | 2023-08-18T20:08:33Z | |
| dc.date.issued | 2023 | |
| dc.description | 40 pages | en_US |
| dc.description.abstract | Dihydrofolate reductase (DHFR) is an essential enzyme in the folic acid synthesis pathway and has been the subject of intense study in recent decades. Despite the wide diversity of homologs, research attention has primarily focused on DHFR proteins from a narrow group of organisms and their mutants. In this study we focus on the ability of DHFR to both rescue metabolic function in a knock-out strain and to 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 carry out a broad mutational scan using a library of nearly 1,000 DHFR homologs and 22,000 mutants synthesized using DropSynth gene synthesis. Variant fitness is determined in a multiplex survival assay in an E. coli ∆FolA∆ThyA knockout strain which allows for conditional selection dependent on external supplementation. We have collected quantitative fitness data on 996 homologs and 22,483 mutants of the DHFR gene based on activity both in the presence and absence of inhibitors, in order to reveal sequence-function relationships and understand how correlations between the fitness landscapes vary as a function of 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 sequence-function relationships which drive antibiotic resistance. | en_US |
| dc.description.embargo | 9999 | |
| dc.identifier.orcid | 0000-0002-3540-625X | |
| dc.identifier.uri | https://hdl.handle.net/1794/28753 | |
| dc.language.iso | en_US | |
| dc.publisher | University of Oregon | |
| dc.rights | CC BY-NC-SA 4.0 | |
| dc.subject | Dihydrofolate reductase | en_US |
| dc.subject | Antibiotic resistance | en_US |
| dc.subject | High-throughput gene synthesis | en_US |
| dc.subject | Broad mutational scan | en_US |
| dc.subject | Enzyme fitness | en_US |
| dc.title | Mapping the Sequence-Function Landscape for Antibiotic Resistance in the DHFR Family | |
| dc.type | Thesis/Dissertation |