Efficient multi-gene expression in cell-free droplet microreactors
KAUST DepartmentStructural Biology and Engineering
Computational Bioscience Research Center (CBRC)
Biological and Environmental Science and Engineering (BESE) Division
KAUST Grant NumberFCC/1/1976-21
Permanent link to this recordhttp://hdl.handle.net/10754/676351
MetadataShow full item record
AbstractCell-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.
CitationSierra, A. M. R., Arold, S. T., & Grünberg, R. (2022). Efficient multi-gene expression in cell-free droplet microreactors. PLOS ONE, 17(3), e0260420. https://doi.org/10.1371/journal.pone.0260420
SponsorsWe thank David Conchouso for critical help with the setup of microfluidics as well as Daniela A. Garcia-Soriano and Marc Guëll for discussions and practical advice. We thank Lyazzat Bekish for purifying the LSSmOrange fluorescent protein. We are grateful to the KAUST Catalysis Center (KCC) and Ulrich Buttner, from the KAUST Nanofabrication Corelab, for providing materials and space for our microfluidic set-up. The KAUST Imaging and Characterization Core lab kindly provided training and assistance for confocal microscopy
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)
PublisherPublic Library of Science (PLoS)
PubMed Central IDPMC8936439
RelationsIs Supplemented By:
Except where otherwise noted, this item's license is described as Archived with thanks to PLOS ONE under a Creative Commons license, details at: http://creativecommons.org/licenses/by/4.0/
- A multifunctional microfluidic platform for generation, trapping and release of droplets in a double laminar flow.
- Authors: Carreras MP, Wang S
- Issue date: 2017 Jun 10
- Selective cell encapsulation, lysis, pico-injection and size-controlled droplet generation using traveling surface acoustic waves in a microfluidic device.
- Authors: Mutafopulos K, Lu PJ, Garry R, Spink P, Weitz DA
- Issue date: 2020 Nov 7
- Multifunctional picoliter droplet manipulation platform and its application in single cell analysis.
- Authors: Gu SQ, Zhang YX, Zhu Y, Du WB, Yao B, Fang Q
- Issue date: 2011 Oct 1
- Droplet CAR-Wash: continuous picoliter-scale immunocapture and washing.
- Authors: Doonan SR, Lin M, Bailey RC
- Issue date: 2019 Apr 23
- Optically-controlled closable microvalves for polymeric centrifugal microfluidic devices.
- Authors: Woolf MS, Dignan LM, Lewis HM, Tomley CJ, Nauman AQ, Landers JP
- Issue date: 2020 Apr 21