Biology subjects

Hinton, S. R.

Publications and source records attributed to Hinton, S. R..

2 recordsLinked to original sources

bioRxiv2025

Exploring Antibiotic Resistance in Diverse Homologs of the Dihydrofolate Reductase Protein Family through Broad Mutational Scanning

Current antibiotic resistance studies often focus on individual protein variants, neglecting broader protein family dynamics. Dihydrofolate reductase (DHFR), a key antibiotic target, has been extensively studied using deep mutational scanning, yet resistance mechanisms across this diverse protein family remain poorly understood. Using DropSynth, a scalable gene synthesis platform, we designed a library of 1,536 synthetic DHFR homologs representing 778 species of bacteria, archaea, and viruses, including clinically relevant pathogens. A multiplexed in vivo assay tested their ability to restore metabolic function and confer trimethoprim resistance in an E. coli {Delta}folA strain. Over half of the synthetic homologs rescued the phenotype without supplementation, and mutants with up to five amino acid substitutions increased the rescue rate to 90%, highlighting DHFRs evolutionary resilience. Broad Mutational Scanning (BMS) of homologs and 100,000 mutants provided critical insights into DHFRs fitness landscape and resistance pathways, representing the most extensive analysis of homolog complementation and inhibitor tolerance to date and advancing our understanding of antibiotic resistance mechanisms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/634126v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@165b786org.highwire.dtl.DTLVardef@f3cc77org.highwire.dtl.DTLVardef@1f4b2c3org.highwire.dtl.DTLVardef@432a6_HPS_FORMAT_FIGEXP M_FIG C_FIG TeaserDropSynth technology enables scalable and cost-effective exploration of antibiotic resistance across the DHFR protein family.

synthetic biology↗

bioRxiv2023

Degenerate DropSynth for Simultaneous Assembly of Diverse Gene Libraries and Local Designed Mutants

Protein engineering efforts often involve the creation of hybrid or chimeric proteins, where functionality critically hinges on the precise design of linkers and fusion points. Traditional methods have been constrained by a focus on single genes or the random selection of fusion points. Here we introduce an approach which enables the creation of large gene libraries where each library comprises a multitude of diverse, specifically designed genes, each with a corresponding set of programmatically designed fusion points or linkers. When combined with multiplex functional assays, these libraries facilitate the derivation of generalized engineering principles applicable across whole protein families or domain types. Degenerate DropSynth is a multiplex gene synthesis technique which allows for the assembly of up to eight distinct variants for each of the 1,536 designed parent genes in a single reaction. We assemble chimeric sensor histidine kinases and demonstrate the assembly of genes up to 1 kbp in length with an 8% rate of perfect assemblies per gene. Our findings indicate that incorporating an increased number of variants in droplets containing barcoded beads does not significantly affect the rate of perfect assemblies. However, maintaining a consistent level of degeneracy across the library is important to ensure good coverage and reduce inequality. The results suggest the potential for scaling this process to assemble at least 8,000 distinct variants in a single reaction. Degenerate DropSynth enables the systematic exploration of protein families through large-scale, programmable assembly of chimeric proteins, moving beyond the limitations of individual protein studies.

synthetic biology↗