Ribozyme and DNA-binding protein expression in emulsion produced by a simple flow-focusing device

Recently, many studies requiring high-throughput screening of large libraries 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 large glass capillary for oil phase, smaller glass capillary for water phase and a thin steel needle for emulsification connected to the exit capillary. Cell-free transcription reaction was performed in emulsion produced by this device. Light mineral oil containing Sun Soft No. 818SK 4% (wt/vol.) and Span 80 1% (wt/vol) was used as the oil phase. Emulsion droplets with diameter around 75 µm were produced.

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 another experiment the single-chain derivative of the lambda Cro protein was expressed in E. coli extract for cell-free protein synthesis in emulsion produced by flow-focusing device. This protein strongly binds to its recognition site (K_D=2×10^-12 M), which was incorporated in the molecule from which it was expressed. It was then visualized with fluorescently-labeled antibody and analyzed by flow cytometer. In both experiments 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.