Efficient multi-gene expression in cell-free droplet microreactors

Abstract

Cell-free transcription and translation systems promise to accelerate and simplify the engineering of proteins, biological circuits and metabolic pathways. Their encapsulation on microfluidic platforms can generate millions of cell-free reactions in picoliter volume droplets. However, current methods struggle to create DNA diversity between droplets while also reaching sufficient protein expression levels. In particular, efficient multi-gene expression has remained elusive. We here demonstrate that co-encapsulation of DNA-coated beads with a defined cell-free system allows high protein expression while also supporting genetic diversity between individual droplets. We optimize DNA loading on commercially available microbeads through direct binding as well as through the sequential coupling of up to three genes via a solid-phase Golden Gate assembly or BxB1 integrase-based recombineering. Encapsulation with an off-the-shelf microfluidics device allows for single or multiple protein expression from a single DNA-coated bead per 14 pL droplet. We envision that this approach will help to scale up and parallelize the rapid prototyping of more complex biological systems.

Clonal amplification-enhanced gene expression for cell-free directed evolution

“…While RCR has not been combined with gene expression yet, RCA and PCR methods are not fully compatible with cell-free protein synthesis due to cross-inhibition by some components or different incubation temperatures. Therefore, multiple-step workflows have been implemented, which require droplet-based microfluidic handling (Mazutis et al, 2009; Fallah-Araghi et al, 2012; Galinis et al, 2016; Holstein et al, 2021) or bead-display (Diamante et al, 2013; Paul et al, 2013; Plesa et al, 2018; Lindenburg et al, 2020; Restrepo Sierra et al, 2022). Still, a major challenge in cell-free directed evolution is the coupling of DNA amplification from single template copies, gene expression and quantitation of the activity of the protein of interest for fitness assignment in one environment.…”