In vitro compartmentalization
Emulsions of cell-like compartments were formed by adding in vitro transcription/translation reaction mixture to stirred mineral oil containing surfactants.
The 3’ of each DNA sequences was purposely designed to contain a HaeIII recognition site which, in the presence of expressed methyltransferase, would be methylated and, thus, resistant to restriction enzyme digestion.
[4] The aqueous phase containing transcription and/or translation components is slowly added to the oil surfactants, and the formation of w/o is facilitated by homogenizing, stirring or using hand extruding device.
The emulsion is quite diverse, and greater homogenization speeds helps to produce smaller droplets with narrower size distribution.
IVC enables the miniaturization of large-scale techniques that can now be done on the micro scale including coupled in vitro transcription and translation (IVTT) experiments.
[9][10] This droplet technology allows high throughput analysis with many different selection pressures in a single experimental setup.
[6][10] IVTT in microdroplets is preferred when overexpression of a desired protein would be toxic to a host cell minimizing the utility of the transcription and translation mechanisms.
[11] IVC has used bacterial cell, wheat germ and rabbit reticulocyte (RRL) extracts for transcription and translation.
Instead, a novel emulsion formulation: 4% Abil EM90 / light mineral oil was developed and demonstrated to be functional in expressing luciferase and human telomerase.
[12] Once transcription and/or translation has completed in the droplets, emulsion will be broken by successive steps of removing mineral oil and surfactants to allow for subsequent selection.
[13] The first method is to attach each DNA molecule with a biotin group and an additional coding sequence for streptavidin (STABLE display).
As an example, Griffiths et al. was able to select for phosphotriesterase variants with higher Kcat by detecting product formation and amount using anti-product antibody and flow cytometry respectively.
1) In an in vivo system, a library of genes is transformed into bacteria to produce the protein variants and dispersed into a water‐in‐oil (w/o) emulsion aiming to have each droplet contain maximum one cell.
2) In an in vitro system, no cells are used and instead protein variants are produced via in vitro transcription/translation aiming to have one gene per droplet.
3,4) In both cases, droplets are then resuspended in water to make a water‐in‐oil‐in‐water (w/o/w) emulsion, have a fluorogenic substrate added, and passed in front of the laser and detector in a fluorescence activated cell sorter. This causes genes that encode active protein variants to be isolated and amplified, sequenced and used for further rounds of selection or directed evolution. [ 2 ]