A Droplet Microfluidic Platform for Automating Genetic Engineering | AIChE

A Droplet Microfluidic Platform for Automating Genetic Engineering

Authors 

Gach, P. - Presenter, Joint BioEnergy Institute
Shih, S. C. C., Joint BioEnergy Institute
Hillson, N. J., DOE Joint BioEnergy Institute
Adams, P. D., Joint BioEnergy Institute
Singh, A. K., Joint Bioenergy Institute

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 E. 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 S. cerevisiae and A. niger. Duration and temperatures of the microfluidic heat-shock procedures where optimized to yield transformation efficiencies comparable to those obtained by bench-top 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.