Recently, high-throughput screening of large libraries of macromolecules like proteins and RNAs are done using water-in-oil emulsion droplets. Microfluidics allows producing uniform emulsion which is beneficial for quantitative assays. We have developed a simple, inexpensive flow-focusing device consisting of two syringes driven by syringe pumps, standard fittings and tubes and a specially fabricated nozzle. This nozzle is made of a larger glass capillary for oil phase, smaller glass capillary for water phase and a thin steel needle for emulsification connected to the exit capillary. The devise was used for cell-free transcription reaction in emulsion. Emulsion droplets with diameter around 75 µm were produced with light mineral oil containing Sun Soft No. 818SK 4% (wt/vol.) and Span 80 1% (wt/vol). An active class I ligase ribozyme bcI-23 was successfully synthesized by T7 RNA-polymerase from DNA attached to paramagnetic beads in the emulsion droplets. This ribozyme ligates itself with RNA sequence attached to the beads thus creating genotype-phenotype linkage. It was then visualized by hybridization with a fluorescently-labeled oligonucleotide and analyzed by flow cytometer. The difference in signal strength between active and inactive promoter variants was much higher (22 times) transcribed in emulsion from flow-focusing device than that obtained in vortex-emulsified reaction (6 times). In addition, the device was used to make emulsion for cell-free protein synthesis reaction. A single-chain derivative of the lambda Cro protein was expressed in an E. coli extract in emulsion produced by the flow-focusing device. This protein strongly binds to its recognition site (KD=2×10-12 M), which was placed on the same beads of the expressed protein. It was then visualized with fluorescently-labeled antibody and analyzed by flow cytometer. In both reactions in emulsion, usage of the flow-focusing device greatly decreased coefficient of variation between the beads with same genotype, showing that it is beneficial for quantitative analysis in high-throughput screening.

In vitro transcription/translation in emulsion produced by a simple flow-focusing device

249th National Meeting (2015)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.