Optimizing the Violacein Biosynthetic Pathway Using Droplet Microfluidics

Gach, P., Joint BioEnergy Institute
Hillson, N. J., Joint BioEnergy Institute
Adams, P. D., Joint BioEnergy Institute
Singh, A. K., Joint BioEnergy Institute
Keasling, J., Lawrence Berkeley National Laboratory
Deutsch, S., DOE Joint Genome Institute
Nath, S., DOE Joint Genome Institute, Lawrence Berkeley National Laboratory
Kaplan, N., Joint BioEnergy Institute
Raje, M., Joint BioEnergy Institute

Synthetic biology experiments require optimization of pathways consisting of many genes and other genetic elements and given the large number of alternatives available for each element, optimization of a pathway can require large number of experiments. Currently, these experiments are done using fairly large amounts of costly reagents per experiment making the process very expensive, extremely slow and irreproducible. Our lab has previously developed droplet-based microfluidic systems for automating specific procedures of synthetic biology: gene assembly and electroporation [1] or heat-shock and culture [2]. However, these operations were performed separately and required additional manual sample preparation to perform the proof-of-principle experiments.

The current system integrates these processes to provide a programmable device for optimizing biosynthetic pathways. We applied this platform to screen for the production of violacein, a molecule with applications in textiles, agriculture and found to have antibacterial, antiviral and anticancer behaviors [3]. Violacein can be biosynthesized in E. coli from L-tryptophan in a process using five enzymes [4]. Digital microfluidics was initially used for dispensing and mixing the gene variants, vector backbone and assembly reagents because of the technologies great control of individual droplets and programmability. This component of the device is capable of generating a combinatorial library composed of 25 variants of the 11 kB violacein producing plasmid VioABCDE. Following combinatorial mixing of gene parts the droplets were transferred to a flow-based PDMS channel for all additional procedures: addition of competent cells, electroporation, addition of culture media, culture, fluorescence imaging and sorting.

Thermal regulation across the system was controlled by positioning peltier modules below the device to provide the 4oC, 37oC, 21oC and 50oC temperatures required for cell storage, cell culture and DNA assembly. Following droplet merger the gene parts were assembled at 21oC for Golden-Gate and 50oC for Gibson assembly, respectively. Gene delivery into electrocompetent cells was then accomplished by providing two 200 ms pulses of 1800 V/ cm by the same electrodes employed for digital microfluidics. Following addition of culture media and 24 hrs culture, successfully transformed cells exhibited a purple color and strong red autofluorescence, indicative of the production of violacein. The flexibility of digital microfluidics, PDMS valves and peltier modules affords quick optimization of operating protocols. The merger of these technologies provides us with a platform, which can perform a variety of operations with minimal electrical and fluidic inputs. This technology will be of great utility for systematic interpretation of gene delivery methods and high-throughput screening of gene variants with minimal reagent requirements.

1. S. C. Shih, G. Goyal, P. W. Kim, N. Koutsoubelis, J. D. Keasling, P. D. Adams, N. J. Hillson and A. K. Singh, ACS Synth Biol, 4(10) 2015, 1151-1164.

2. P. C. Gach, S. C. Shih, J. Sustarich, J. D. Keasling, N. J. Hillson, P. D. Adams and A. K. Singh, ACS Synth Biol, 5(5) 2016, 426-433.

3. S. Y. Choi, K. H. Yoon, J. I. Lee and R. J. Mitchell, Biomed Res Int, 2015, 2015, 465056.

4. C. J. Balibar and C. T. Walsh, Biochemistry, 2006, 45, 15444-15457.