Adhesive Tape Microfluidics with an Autofocusing Module That Incorporates CRISPR Interference: Applications to Long-Term Bacterial Antibiotic Studies

Abstract

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.

Rapid Production of PDMS Microdevices for Electrodriven Separations and Microfluidics by 3D-Printed Scaffold Removal

“…Our setup does not need any highly expensive way of production, and it can be used for a few purposes. There are some other low-cost preparation methods, including laser cutting and scribing adhesive tape [31][32][33]. However, lasers are still more expensive.…”

Impact of CRISPR interference on strain development in biotechnology

“…Successful Gfp fluorescence silencing represents a new ability to repress genes of interest in B. apis , enabling functional studies of essential genes that cannot be disrupted entirely [85]. Furthermore, CRISPRi was used in E. coli to record morphological changes during ampicillin exposure in microfluidic platforms and to deduce the minimum inhibitory concentration of this antibiotic on the single‐cell level [86].…”

Real-time respiration changes as a viability indicator for rapid antibiotic susceptibility testing in a microfluidic chamber array

“…12 This also significantly shortens the time required for pre-culturing and further analysis. Additionally, microfluidic techniques can be combined with molecular ASTs 13,14 facilitating complementary bacterial identification or analysis. 15 Droplet systems offer compartmentalization of the bacterial samples, continuously and in a very high throughput, enabling analysis of thousands of samples 16 and on the single-cell level.…”