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. 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 proposed platform offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput.
authors.affiliation: Technology Division, Joint BioEnergy Institute (JBEI) , Emeryville, California 94608, United States.|Applied Biosciences and Engineering, Sandia National Laboratories , Livermore, California 94550, United States.
authors.forename: Philip C
authors.initials: PC
authors.lastname: Gach
authors.affiliation: Technology Division, Joint BioEnergy Institute (JBEI) , Emeryville, California 94608, United States.|Applied Biosciences and Engineering, Sandia National Laboratories , Livermore, California 94550, United States.
authors.forename: Steve C C
authors.initials: SC
authors.lastname: Shih
authors.affiliation: Technology Division, Joint BioEnergy Institute (JBEI) , Emeryville, California 94608, United States.|Applied Biosciences and Engineering, Sandia National Laboratories , Livermore, California 94550, United States.
authors.forename: Jess
authors.initials: J
authors.lastname: Sustarich
authors.affiliation: Fuels Synthesis Division, Joint BioEnergy Institute (JBEI) , Emeryville, California 94608, United States.|Biological Systems and Engineering Division, Lawrence Berkeley National Lab , Berkeley, California 94720, United States.|Department of Bioengineering, University of California , Berkeley, California 94720, United States.|Department of Chemical & Biomolecular Engineering, University of California , Berkeley, California 94720, United States.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Technology Division, Joint BioEnergy Institute (JBEI) , Emeryville, California 94608, United States.|Fuels Synthesis Division, Joint BioEnergy Institute (JBEI) , Emeryville, California 94608, United States.|Biological Systems and Engineering Division, Lawrence Berkeley National Lab , Berkeley, California 94720, United States.|DOE Joint Genome Institute , Walnut Creek, California 94598, United States.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Hillson
authors.affiliation: Technology Division, Joint BioEnergy Institute (JBEI) , Emeryville, California 94608, United States.|Department of Bioengineering, University of California , Berkeley, California 94720, United States.|Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Lab , Berkeley, California 94720, United States.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.affiliation: Technology Division, Joint BioEnergy Institute (JBEI) , Emeryville, California 94608, United States.|Applied Biosciences and Engineering, Sandia National Laboratories , Livermore, California 94550, United States.
authors.forename: Anup K
authors.initials: AK
authors.lastname: Singh
mesh_terms: D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D044085:Microfluidics / Q000379:methods*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D004247:DNA
keywords: cell culture; digital microfluidics; molecular biology; transformation