Efficient multi-gene expression in cell-free droplet microreactors

PLoS One. 2022 Mar 21;17(3):e0260420. doi: 10.1371/journal.pone.0260420. eCollection 2022.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / genetics
  • Gene Expression
  • Lab-On-A-Chip Devices
  • Microfluidic Analytical Techniques*
  • Microfluidics

Substances

  • DNA

Grants and funding

Funding was provided by the King Abdullah University of Science and Technology (KAUST) through the baseline fund and the Award No. FCC/1/1976-21 from the Office of Sponsored Research (OSR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.