Errors in protein synthesis have historically been assumed to be detrimental to the cell. While there are many reports that translational errors are consequential, there is a growing body of evidence that some mistranslation events may be tolerated or even beneficial. Using two models of mistranslation, we compare the direct phenotypic effects of these events in Escherichia coli. This work provides insight into the threshold for tolerance of specific mistranslation events that were previously predicted to be broadly neutral to proteome integrity. Furthermore, these data reveal the effects of mistranslation beyond the general unfolded stress response, leading to global translational reprogramming.

The ability to study bacteria at the single cell level has advanced our insights into microbial physiology and genetics in ways not attainable by studying large populations using more traditional culturing methods. To improve methods to characterize bacteria at the cellular level, we developed a new microfluidic platform that enables cells to be exposed to metabolites in a gradient of concentrations. By designing lowcost, three-dimensional devices with adhesive tapes and tailoring them for bacterial imaging, we avoided the complexities of silicon and polymeric microfabrication. The incorporation of an agarose membrane as the resting substrate, along with a temperature-controlled environmental chamber, allows the culturing of bacterial cells for over 10 h under stable growth or inhibition conditions. Incorporation of an autofocusing module helped the uninterrupted, high-resolution observation of bacteria at the single-cell and at low density population levels. We used the microfluidic platform to record morphological changes in Escherichia coli during ampicillin exposure and to quantify the minimum inhibitory concentration of the antibiotic. We further demonstrated the potential of finely-tuned, incremental gene regulation in a concentration gradient utilizing CRISPR interference (CRISPRi). These low-cost engineering tools, when implemented in combination with genetic approaches such as CRISPRi, should prove useful to uncover new genetic determinants of antibiotic susceptibility and evaluate the long-term effectiveness of antibiotics in bacterial cultures.

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New combinations of existing antibiotics are being investigated to combat bacterial resilience. This requires detection technologies with reasonable cost, accuracy, resolution, and throughput. Here, we present a multi-drug screening platform for bacterial cultures by combining droplet microfluidics, search algorithms, and imaging with a wide field of view. We remotely alter the chemical microenvironment around cells and test 12 combinations of resistant cell types and chemicals. Fluorescence intensity readouts allow us to infer bacterial resistance to specific antibiotics within 8 hours. The platform has potential to detect and identify parameters of bacterial resilience in cell cultures, biofilms, and microbial aggregates.

Over the last decade, the study of CRISPR-Cas systems has progressed from a newly discovered bacterial defense mechanism to a diverse suite of genetic tools that have been applied across all domains of life. While the initial applications of CRISPR-Cas technology fulfilled a need to more precisely edit eukaryotic genomes, creative “repurposing” of this adaptive immune system has led to new approaches for genetic analysis of microorganisms, including improved gene editing, conditional gene regulation, plasmid curing and manipulation, and other novel uses.