J.A. Wippold, M.J. Chu, B.L. Adams, A. Han
U.S. Army Combat Capabilities Development Command – Army Research Laboratory, SynBio Tools Branch, Biology and Biological Sciences Division, Army Research Directorate,
United States
Keywords: droplet microfluidics, synthetic biology, gene transfer, high-throughput screening
Summary:
A significant hurdle for the widespread implementation of synthetic biology tools into various microbes is the challenge of high throughput DNA introduction into cells. Common approaches to DNA introduction into cells, such transformation and electroporation, are typically only applicable to a small subset of laboratory bacterial strains and thus leave the vast majority of microbial life genetically inaccessible. To address this challenge, we extended the MIT developed cell-to-cell DNA tool XPORT into a high throughput microfluidic system. DNA ENTRAP (DNA ENhanced TRAnsfer Platform) is a novel droplet-based microfluidic platform that generates nano-bioreactors, providing the capability to streamline the development of genetic transfer. Droplet microfluidic technology has demonstrated utility as a screening mechanism for numerous biological applications using droplet-encapsulated microbes. We demonstrate DNA ENTRAP simplifies, enhances, and increases the throughput of XPORT-mediated conjugation, cell-to-cell DNA transfer using microfluidics. We designed the platform to perform the following functions: generate a large number of nano-liter scale bioreactors encapsulating cell candidates; incubate cell-encapsulated droplets for varying times; modular control over cellular encapsulation; and adaptability to future pairing with automation. On-chip viability assays confirmed donor and recipient strains viability (< 3% death). The team tested the underlying technology (XPORT conjugation) of DNA ENTRAP at two donor to recipient cell ratios 3:1 & 1:1, respectively, for 0.5 hr, 1 hr, and 2 hr co-incubation periods. We determined the conjugation efficiency to be superior (2 – 20-fold increase) when using DNA ENTRAP compared to the conventional benchtop method at every tested condition. Genome sequencing and FACS further confirmed the on-chip results and an application demonstration was completed using an example cargo gene. DNA ENTRAP will be an essential tool in the future synthesis, discovery, and development of novel organisms with potential applications in military, therapeutics, and energy.