Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption.
Phenotyping single cells based on the products they secrete or consume is a key bottleneck in many biotechnology applications, such as combinatorial metabolic engineering for the overproduction of secreted metabolites. Here we present a flexible high-throughput approach that uses microfluidics to compartmentalize individual cells for growth and analysis in monodisperse nanoliter aqueous droplets surrounded by an immiscible fluorinated oil phase. We use this system to identify xylose-overconsuming Saccharomyces cerevisiae cells from a population containing one such cell per 10(4) cells and to screen a genomic library to identify multiple copies of the xylose isomerase gene as a genomic change contributing to high xylose consumption, a trait important for lignocellulosic feedstock utilization. We also enriched L-lactate-producing Escherichia coli clones 5,800× from a population containing one L-lactate producer per 10(4) D-lactate producers. Our approach has broad applications for single-cell analyses, such as in strain selection for the overproduction of fuels, chemicals and pharmaceuticals.
1] Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2].
Benjamin L
BL
Wang
1] Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2].
Adel
A
Ghaderi
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Hang
H
Zhou
Department of Physics and School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA.
Jeremy
J
Agresti
Department of Physics and School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA.
David A
DA
Weitz
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
Gerald R
GR
Fink
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Gregory
G
Stephanopoulos
D004926:Escherichia coli / Q000166:cytology / Q000302:isolation & purification; D005110:Extracellular Space / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D019344:Lactic Acid / Q000032:analysis / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000302:isolation & purification; D059010:Single-Cell Analysis / Q000379:methods*
D019344:Lactic Acid