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Nat Commun. 2017 Aug 23;8(1):332. doi: 10.1038/s41467-017-00425-7.

RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes.

Author information

1
Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St Stop C0400, Austin, Texas, 78712, USA.
2
Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, 94158, California, USA.
3
California Institute for Quantitative Biosciences, University of California San Francisco, San Francisco, 94158, California, USA.
4
Chan Zuckerberg Biohub, San Francisco, 94158, California, USA.
5
Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, Texas, 78712, USA.
6
Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St Stop C0400, Austin, Texas, 78712, USA. halper@che.utexas.edu.
7
Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, Texas, 78712, USA. halper@che.utexas.edu.
8
Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, 94158, California, USA. adam@abatelab.org.
9
California Institute for Quantitative Biosciences, University of California San Francisco, San Francisco, 94158, California, USA. adam@abatelab.org.
10
Chan Zuckerberg Biohub, San Francisco, 94158, California, USA. adam@abatelab.org.

Abstract

Synthetic biology and metabolic engineering seek to re-engineer microbes into "living foundries" for the production of high value chemicals. Through a "design-build-test" cycle paradigm, massive libraries of genetically engineered microbes can be constructed and tested for metabolite overproduction and secretion. However, library generation capacity outpaces the rate of high-throughput testing and screening. Well plate assays are flexible but with limited throughput, whereas droplet microfluidic techniques are ultrahigh-throughput but require a custom assay for each target. Here we present RNA-aptamers-in-droplets (RAPID), a method that greatly expands the generality of ultrahigh-throughput microfluidic screening. Using aptamers, we transduce extracellular product titer into fluorescence, allowing ultrahigh-throughput screening of millions of variants. We demonstrate the RAPID approach by enhancing production of tyrosine and secretion of a recombinant protein in Saccharomyces cerevisiae by up to 28- and 3-fold, respectively. Aptamers-in-droplets affords a general approach for evolving microbes to synthesize and secrete value-added chemicals.Screening libraries of genetically engineered microbes for secreted products is limited by the available assay throughput. Here the authors combine aptamer-based fluorescent detection with droplet microfluidics to achieve high throughput screening of yeast strains engineered for enhanced tyrosine or streptavidin production.

PMID:
28835641
PMCID:
PMC5569033
DOI:
10.1038/s41467-017-00425-7
[Indexed for MEDLINE]
Free PMC Article

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