Bacterial Microcolonies in Gel Beads for High-throughput Screening.

Bio-protocol
Yolanda Schaerli

Abstract

High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants. Here, I describe a protocol for high-throughput screening of bacterial (E. coli) microcolonies in gel beads. Single cells are encapsulated into monodisperse water-in-oil emulsion droplets produced with a microfluidic device. The aqueous solution also contains agarose that gelates upon cooling on ice, so that solid gel beads form inside the droplets. During incubation of the emulsion, the cells grow into monoclonal microcolonies inside the beads. After isolation of the gel beads from the emulsion and their sorting by fluorescence activated cell sorting (FACS), the bacteria are recovered from the gel beads and are then ready for a further round of sorting, mutagenesis or analysis. In order to sort by FACS, this protocol requires a fluorescent readout, such as the expression of a fluorescent reporter protein. Measuring the average fluorescent signals of microcolonies reduces the influence of hig...Continue Reading

References

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Aug 22, 2014·Nature Chemistry·Martin FischlechnerFlorian Hollfelder
Aug 15, 2017·ACS Synthetic Biology·José M DuarteYolanda Schaerli

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Citations

Dec 7, 2019·Analytical Chemistry·Malgorzata A WitekSteven A Soper

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