Figure 1: A schematic workflow for screening high-affinity binders using drop-based in vitro two-hybrid method (drop-IVT2H).
From: A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders
(A) Single DNA templates and IVT2H reagents were encapsulated in drops on a microfluidic chip by applying vacuum to generate monodisperse drops. The binder DNA template was distributed as single copy in drops based on Poisson statistics. Each drop contained multiple copies of the reporter and target DNA templates. Drops were then collected for off-chip incubation. Inset, the IVT2H reaction during off-chip incubation. PMI-DB and AD-MDM2 were expressed from the binder and target templates, respectively. PMI-DB bound the upstream activation sequence (UAS) on the reporter DNA. The interaction of PMI and MDM2 recruited AD to the promoter-bound RNA polymerase (RNAP) and activated the expression of the reporter gene, producing the fluorescent GFP. (B) After incubation, drops were re-injected into a fluorescence-activated drop sorting device (FADS). Co-flowing spacing oil ensured equal separation of drops. Both bright and dark drops were isolated and collected in separate tubes for RT-PCR followed by Sanger or deep sequencing.
