Agarose-based microwell array chip for high-throughput screening of functional microorganisms

Abstract

Natural bacterial communities provide a rich source of biocatalysts, however, high-throughput screening for the functional bacteria remains a major challenge. Here, we present an agarose-based microwells array chip for functionally screening and isolating novel microorganisms with merits of high-throughput, high specificity and sensitivity. In this approach, the bacterial cells were loaded with single cell per a microwell mode and were incubated in the arrayed agarose microwells. The growths of single cells were then monitored in real time and the enzyme reaction activities were assessed at the individual microwell resolution. To validate the reliability of the method, we subsequently applied it to screen lipase-producing bacteria from the pond water based on lipase hydrolysis of the substrate in the presence of a fluorescent dye (rhodamine B), which emitted yellow-orange fluorescent halos upon UV-light irradiation. A total amount of more than 310,000 isolates from pond water could be screened at a time in only 13 h with reduced consumption of reagents. As a result, 12 microcolonies were identified out with the desired phenotype, in which two new species were discovered based on 16S rRNA sequencing. We expect the developed method to be potentially useful to high-throughput analysis, microbiology and synthetic biology.

Keywords: High-throughput screening; Lipase; Microorganism; Microwells array.