A Multiplexed CRISPR Interference Approach to Investigate the Genetic Interactions for Spectinomycin Antibiotic Resistance and Biofilm Formation in Escherichia coli

Presenter

Dominic Castaldi

Campus

UMass Amherst

Sponsor

Lauren B. Andrews, Department of Chemical Engineering, UMass Amherst

Schedule

Session 4, 2:30 PM - 3:15 PM [Schedule by Time][Poster Grid for Time/Location]

Location

Poster Board A40, Campus Center Auditorium, Row 2 (A21-A40) [Poster Location Map]

Abstract

Antibiotic resistance is a global health threat, exacerbated by bacterial biofilms that exhibit heightened resistance. Escherichia coli is a leading cause of antibiotic-resistant deaths and presents a research gap in understanding the genetic mechanisms behind antibiotic resistance. This study aims to identify the genetic links between antibiotic resistance and biofilm formation using CRISPR interference (CRISPRi) gene silencing for a model antibiotic, spectinomycin. We applied a genome-wide CRISPRi library of E. coli K-12 MG1655 previously constructed by our group to identify gene targets that confer the phenotypical responses. The library contains 34,347 designed gRNA targeting >99.9% of all protein and RNA gene products. We first assayed and determined the antibiotic MIC₅₀, the concentration for a 50% reduction in cell growth, using cell growth kinetic experiments. These experiments were completed for the E. coli MG1655 wildtype strain with and without the CRISPRi destination plasmid under aerobic growth conditions in a 96-well microtiter plate with the addition of varying concentrations of spectinomycin. We observed that both bacterial strains had a 50% reduction in growth rate with 40 μg/mL spectinomycin. Next, we conducted selections with the genome-wide CRISPRi library and 40 μg/mL spectinomycin to identify genes associated with spectinomycin resistance. The pooled CRISPRi cell library was grown alongside the wildtype strain to compare growth to natural adaptation by laboratory evolution. Each culture was passaged in 24-hour intervals under aerobic conditions. Further experiments will analyze the pooled samples showing increased growth by DNA sequencing to determine the gRNAs and gene targets associated with spectinomycin resistance.

Keywords

Synthetic Biology, Metabolic Engineering, Genetic Engineering

Research Area

Biological Organisms

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