Bacterial Microcolonies in Gel Beads for High-throughput Screening.

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Version: Final published version
License: CC BY 4.0
Serval ID
serval:BIB_E06DF5436657
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Bacterial Microcolonies in Gel Beads for High-throughput Screening.
Journal
Bio-Protocol
Author(s)
Schaerli Y.
ISSN
2331-8325 (Print)
ISSN-L
2331-8325
Publication state
Published
Issued date
05/07/2018
Peer-reviewed
Oui
Volume
8
Number
13
Pages
e2911
Language
english
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 ( <i>E. coli</i> ) 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 high phenotypic cell-to-cell variability and increases the sensitivity compared to the sorting of single cells. We applied this method to sort a pBAD promoter library at ON and OFF states (Duarte <i>et al.</i> , 2017).
Keywords
Combinatorial libraries, Directed evolution, Gel beads, High-throughput screening, Microcolonies, Microdroplets, Synthetic biology
Pubmed
Create date
31/07/2018 16:49
Last modification date
30/07/2020 6:21
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