A Droplet Microfluidic Platform for Automating Genetic Engineering

被引:58
作者
Gach, Philip C. [1 ,2 ]
Shih, Steve C. C. [1 ,2 ]
Sustarich, Jess [1 ,2 ]
Keasling, Jay D. [3 ,4 ,5 ,6 ]
Hillson, Nathan J. [1 ,3 ,4 ,7 ]
Adams, Paul D. [1 ,5 ,8 ]
Singh, Anup K. [1 ,2 ]
机构
[1] Joint BioEnergy Inst JBEI, Div Technol, Emeryville, CA 94608 USA
[2] Sandia Natl Labs, Appl Biosci & Engn, Livermore, CA 94550 USA
[3] Joint BioEnergy Inst JBEI, Fuels Synth Div, Emeryville, CA 94608 USA
[4] Univ Calif Berkeley, Lawrence Berkeley Natl Lab, Biol Syst & Engn Div, Berkeley, CA 94720 USA
[5] Univ Calif Berkeley, Dept Bioengn, Berkeley, CA 94720 USA
[6] Univ Calif Berkeley, Dept Chem & Biomol Engn, Berkeley, CA 94720 USA
[7] US DOE, Joint Genome Inst, Walnut Creek, CA 94598 USA
[8] Univ Calif Berkeley, Lawrence Berkeley Natl Lab, Mol Biophys & Integrated Bioimaging Div, Berkeley, CA 94720 USA
来源
ACS SYNTHETIC BIOLOGY | 2016年 / 5卷 / 05期
关键词
digital microfluidics; molecular biology; transformation; cell culture; DIGITAL MICROFLUIDICS; ESCHERICHIA-COLI; TRANSFORMATION; ASPERGILLUS; BACTERIA; CULTURE; CLONING;
D O I
10.1021/acssynbio.6b00011
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. 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 (similar to 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 proposed platform offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput.
引用
收藏
页码:426 / 433
页数:8
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