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.

A Guide to Biodetection in Droplets

“…Sensitivity of the assay was estimated at 0.05 ng per μL of cDNA for the FKBP5 target gene and 1 copy per μL of a reference gene. To simplify multiprotein expression studies, Sierra et al utilized Streptavidin-coated polystyrene beads for binding/coating target DNA . This enabled the creation of cell-free expression systems inside droplets.…”

“…The gene expression level was detected and quantified via a fluorescence emission signal from biotinylated DNA encoding mScarlet, mNeonGreen, and LSSmOrange fluorescence labels. Overall, this cell-free system eliminates cloning and could potentially accelerate the design–build–test cycle in synthetic biology as reported by Sierra et al Droplet encapsulation coupled with fluorescence detection can also enhance detection and expression level measurement of specific target mRNA transcripts from single cells, as exemplified by the recent works of Hyman et al The invented droplet platform is called single-cell nucleic acid profiling in droplets (SNAPD) and was used for analyzing transcriptional biomarkers in thousands of single mammalian cells. By incorporating LAMP amplification and multiple universal labels such as FAM, HEX, and SYBR green, Hyman et al could perform a multiplex assay where fluorescence signals were based on specific target gene sequences of the biomarkers .…”

Clonal Amplification-Enhanced Gene Expression in Synthetic Vesicles

“…Thus, so far, DNA templates cannot be amplified efficiently in the same solution where the cell-free system is performed. Therefore, multiple-step workflows have been implemented, which require droplet-based microfluidic handling ,− or bead display. − …”

“…Therefore, multiple-step workflows have been implemented, which require droplet-based microfluidic handling 28 , 51 − 53 or bead display. 54 − 58 …”

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

“…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.…”