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Title: A Droplet Microfluidic Platform for Automating Genetic Engineering

Abstract

We present a water-in-oil droplet microfluidic platform for transformation, culture and expression of recombinant proteins in multiple host organisms including bacteria, yeast and fungi. The platform consists of a hybrid digital microfluidic/channel-based droplet chip with integrated temperature control to allow complete automation and integration of plasmid addition, heat-shock transformation, addition of selection medium, culture, and protein expression. The microfluidic format permitted significant reduction in consumption (100-fold) of expensive reagents such as DNA and enzymes compared to the benchtop method. Furthermore, the chip contains a channel to continuously replenish oil to the culture chamber to provide a fresh supply of oxygen to the cells for long-term (~5 days) cell culture. The flow channel also replenished oil lost to evaporation and increased the number of droplets that could be processed and cultured. The platform was validated by transforming several plasmids into Escherichia coli including plasmids containing genes for fluorescent proteins GFP, BFP and RFP; plasmids with selectable markers for ampicillin or kanamycin resistance; and a Golden Gate DNA assembly reaction. We also demonstrate the applicability of this platform for transformation in widely used eukaryotic organisms such as Saccharomyces cerevisiae and Aspergillus Niger. Duration and temperatures of the microfluidic heat-shock procedures were optimizedmore » to yield transformation efficiencies comparable to those obtained by benchtop methods with a throughput up to 6 droplets/min. The platform we proposed offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput.« less

Authors:
 [1];  [1];  [1];  [2];  [3];  [4];  [1]
+ Show Author Affiliations
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States). Technology Division; Sandia National Lab. (SNL-CA), Livermore, CA (United States). Applied Biosciences adn Engineering
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States). Fuels Synthesis Division; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering
  3. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States). Technology Division, Fuels Synthesis Division; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division
  4. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States). Technology Division; Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering
Publication Date:
Research Org.:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1379348
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 5; Journal Issue: 5; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; digital microfluidics; molecular biology; transformation; cell culture

Citation Formats

Gach, Philip C., Shih, Steve C. C., Sustarich, Jess, Keasling, Jay D., Hillson, Nathan J., Adams, Paul D., and Singh, Anup K. A Droplet Microfluidic Platform for Automating Genetic Engineering. United States: N. p., 2016. Web. doi:10.1021/acssynbio.6b00011.
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Gach, Philip C., Shih, Steve C. C., Sustarich, Jess, Keasling, Jay D., Hillson, Nathan J., Adams, Paul D., & Singh, Anup K. A Droplet Microfluidic Platform for Automating Genetic Engineering. United States. https://doi.org/10.1021/acssynbio.6b00011
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Gach, Philip C., Shih, Steve C. C., Sustarich, Jess, Keasling, Jay D., Hillson, Nathan J., Adams, Paul D., and Singh, Anup K. Mon . "A Droplet Microfluidic Platform for Automating Genetic Engineering". United States. https://doi.org/10.1021/acssynbio.6b00011. https://www.osti.gov/servlets/purl/1379348.
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@article{osti_1379348,
title = {A Droplet Microfluidic Platform for Automating Genetic Engineering},
author = {Gach, Philip C. and Shih, Steve C. C. and Sustarich, Jess and Keasling, Jay D. and Hillson, Nathan J. and Adams, Paul D. and Singh, Anup K.},
abstractNote = {We present a water-in-oil droplet microfluidic platform for transformation, culture and expression of recombinant proteins in multiple host organisms including bacteria, yeast and fungi. The platform consists of a hybrid digital microfluidic/channel-based droplet chip with integrated temperature control to allow complete automation and integration of plasmid addition, heat-shock transformation, addition of selection medium, culture, and protein expression. The microfluidic format permitted significant reduction in consumption (100-fold) of expensive reagents such as DNA and enzymes compared to the benchtop method. Furthermore, the chip contains a channel to continuously replenish oil to the culture chamber to provide a fresh supply of oxygen to the cells for long-term (~5 days) cell culture. The flow channel also replenished oil lost to evaporation and increased the number of droplets that could be processed and cultured. The platform was validated by transforming several plasmids into Escherichia coli including plasmids containing genes for fluorescent proteins GFP, BFP and RFP; plasmids with selectable markers for ampicillin or kanamycin resistance; and a Golden Gate DNA assembly reaction. We also demonstrate the applicability of this platform for transformation in widely used eukaryotic organisms such as Saccharomyces cerevisiae and Aspergillus Niger. Duration and temperatures of the microfluidic heat-shock procedures were optimized to yield transformation efficiencies comparable to those obtained by benchtop methods with a throughput up to 6 droplets/min. The platform we proposed offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput.},
doi = {10.1021/acssynbio.6b00011},
journal = {ACS Synthetic Biology},
number = 5,
volume = 5,
place = {United States},
year = {Mon Feb 01 00:00:00 EST 2016},
month = {Mon Feb 01 00:00:00 EST 2016}
}
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https://doi.org/10.1021/acssynbio.6b00011
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