Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

Abstract

Protein expression in vitro has broad applications in directed evolution, synthetic biology, proteomics and drug screening. However, most of the in vitro expression systems rely on relatively high DNA template concentrations to obtain sufficient amounts of proteins, making it harder to perform in vitro screens on gene libraries. Here, we report a technique for the generation of condensed DNA particles that can serve as efficient templates for in vitro gene expression. We apply droplet microfluidics to encapsulate single-DNA molecules in 3-picoliter (pL) volume droplets and convert them into 1 μm-sized DNA particles by the multiple displacement amplification reaction driven by phi29 DNA polymerase. In the presence of magnesium ions and inorganic pyrophosphate, the amplified DNA condensed into the crystalline-like particles, making it possible to purify them from the reaction mix by simple centrifugation. Using purified DNA particles, we performed an in vitro transcription-translation reaction and successfully expressed complex enzyme β-galactosidase in droplets and in the 384-well format. The yield of protein obtained from DNA particles was significantly higher than from the corresponding amount of free DNA templates, thus opening new possibilities for high throughput screening applications.

Towards a synthetic cell cycle

“…Before considering division strategies, it is important to briefly consider the pros and cons of different types of synthetic containers. Water-in-oil droplets are an attractive option for testing purposes: very easy to mass-produce through microfluidics 41 and offering excellent encapsulation of components. Yet, the large surface tension makes these droplets difficult to deform, and the external hydrophobic environment hardly allows any transport in and out of the synthetic cell.…”

“…Images depicting droplets, coacervates, lipids and lipid shape transformation, mechanical deformation, FtsZ, and DNA origami are reproduced from refs. 41,42,54,55,57,70 , respectively, under the CC BY 4.0 licence. Images depicting Min system, Min are reproduced/adapted from ref.…”

“…Droplets 41 Liposomes Coacervates 42 ESCRT-III 72 Lipid shape transformaƟon 54 Min system 43 Lipid phase separaƟon 51 Microtubules Mechanical spliƫng 108 AcƟve droplets 74 Min 43 Lipids 54…”

Fast and Ample Light Controlled Actuation of Monodisperse All‐DNA Microgels

“…[23,24] When combined to microfluidics, the structures are highly controlled as exemplified with DNA solutions producing liquid micro-compartments [25] or with the preparation of crystalline particles of clonal DNA sequences. [26,27] We recently combined DNA liquid-liquid phase separation and supramolecular hybridization to explore new assembly landscapes for the pathway controlled formation of mesoscale all-DNA microgels. [28] However, the arrested phase separation at the origin of these DNA colloids yielded highly polydisperse particles, which prevented their assembly into well-defined superstructures.…”

Viscosity-Regulated Control of RNA Microstructure Fabrication

“…The inorganic pyrophosphate, Mg 2 PPi, is a biocompatible compound and a naturally occurring biological molecule [ 10 ]. Mg 2 PPi precipitate out from the solution and form crystalline structures with the adsorption of produced RNA and finally assemble into a unique sponge-like structure [ 11 , 12 , 13 ]. Specifically, RNA acts as a nucleation promoter at the initial stage and an inhibitor of crystal growth at the later stage through the interaction of RNA with the crystal surface.…”