Can Altered Bioenergetics Drive Antibiotic Persistence in Low-Oxygen Conditions?
Abstract
The goal of this project is to engineer the first set of anaerobically visible fluorescent ATP indicators and apply them to examine how intracellular bioenergetics differs between aerobic and anaerobic states for bacterial cells treated with antibiotics. While several ATP sensors are currently available, these require oxygen to produce fluorescence. To address this issue, we propose to develop our sensors based on fluorescent proteins that do not require oxygen. In this project period, we established the key conceptual and methodological framework for constructing these sensors. Significant findings include: (1) identification of 14 viable positions in the reporter backbone where binding domains may be inserted to modulate fluorescence properties based on the principle of allostery (2) establishing a linker mutagenesis and screening pipeline for high-throughput testing of candidate sensor constructs for ATP-induced changes in fluorescence (3) validation of methods for titrating intracellular ATP without causing changes in overall reporter fluorescence (4) identification of terminal amino acids that may be truncated to shorten overall size of the reporter construct by 12 amino acids (potentially making this the smallest reporter gene currently available).
Document Details
- Document Type
- Technical Report
- Publication Date
- Mar 01, 2021
- Accession Number
- AD1139942
Entities
People
- Arnab Mukherjee
Organizations
- University of California