Smart DNA Fabrication Using Sound Waves

Acoustic droplet ejection (ADE) technology uses focused acoustic energy to transfer nanoliter-scale liquid droplets with high precision and accuracy. This noncontact, tipless, low-volume dispensing technology minimizes the possibility of cross-contamination and potentially reduces the costs of reagents and consumables. To date, acoustic dispensers have mainly been used in screening libraries of compounds. In this paper, we describe the first application of this powerful technology to the rapidly developing field of synthetic biology, for DNA synthesis and assembly at the nanoliter scale using a Labcyte Echo 550 acoustic dispenser. We were able to successfully downscale PCRs and the popular one-pot DNA assembly methods, Golden Gate and Gibson assemblies, from the microliter to the nanoliter scale with high assembly efficiency, which effectively cut the reagent cost by 20- to 100-fold. We envision that acoustic dispensing will become an instrumental technology in synthetic biology, in particular in the era of DNA foundries.

[1]  Timothy S. Ham,et al.  Production of the antimalarial drug precursor artemisinic acid in engineered yeast , 2006, Nature.

[2]  Joel S. Bader,et al.  Synthetic chromosome arms function in yeast and generate phenotypic diversity by design , 2011, Nature.

[3]  X. Chen,et al.  Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Richard N. Ellson,et al.  Transfer of low nanoliter volumes between microplates using focused acoustics-automation considerations , 2003 .

[5]  D. Endy.  Foundations for engineering biology , 2005, Nature.

[6]  Philip Ball,et al.  Synthetic biology: Starting from scratch , 2004, Nature.

[7]  Farren J. Isaacs,et al.  Recoded organisms engineered to depend on synthetic amino acids , 2015, Nature.

[8]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[9]  Stephen R. Quake,et al.  A microfluidic oligonucleotide synthesizer , 2010, Nucleic acids research.

[10]  D A. Dunn,et al.  Challenges and solutions to ultra-high-throughput screening assay miniaturization: submicroliter fluid handling. , 2000, Drug discovery today.

[11]  Alistair Elfick,et al.  PaperClip: rapid multi-part DNA assembly from existing libraries , 2014, Nucleic acids research.

[12]  Drew Endy,et al.  Precise and reliable gene expression via standard transcription and translation initiation elements , 2013, Nature Methods.

[13]  Yizhi Cai,et al.  Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes , 2015, Proceedings of the National Academy of Sciences.

[14]  Swapnil Bhatia,et al.  Developer's and user's guide to Clotho v2.0 A software platform for the creation of synthetic biological systems. , 2011, Methods in enzymology.

[15]  Nathan J Hillson,et al.  j5 DNA assembly design automation software. , 2012, ACS synthetic biology.

[16]  Carola Engler,et al.  Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes , 2009, PloS one.

[17]  J. Collins,et al.  A brief history of synthetic biology , 2014, Nature Reviews Microbiology.

[18]  W. Edelmann,et al.  SLiCE: a novel bacterial cell extract-based DNA cloning method , 2012, Nucleic acids research.

[19]  M. Itaya,et al.  Combining two genomes in one cell: stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Judy Qiu,et al.  Total Synthesis of a Functional Designer Eukaryotic Chromosome , 2014, Science.

[21]  Yoke Kong Kuan,et al.  Integrated two-step gene synthesis in a microfluidic device. , 2009, Lab on a chip.

[22]  Timothy B. Stockwell,et al.  Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome , 2008, Science.

[23]  Jingdong Tian,et al.  Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways , 2009, PloS one.

[24]  Ryo Takeuchi,et al.  Biocontainment of genetically modified organisms by synthetic protein design , 2015, Nature.

[25]  David S. Kong,et al.  Parallel gene synthesis in a microfluidic device , 2007, Nucleic acids research.

[26]  Ron Weiss,et al.  Engineering life: building a fab for biology. , 2006, Scientific American.

[27]  A. Paul,et al.  Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template , 2002, Science.

[28]  James J. Collins,et al.  Paper-Based Synthetic Gene Networks , 2014, Cell.

[29]  Thomas H Segall-Shapiro,et al.  Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome , 2010, Science.

[30]  Farren J. Isaacs,et al.  Multilayered genetic safeguards limit growth of microorganisms to defined environments , 2015, Nucleic acids research.

[31]  Timothy K Lu,et al.  Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy , 2009, Proceedings of the National Academy of Sciences.

[32]  Olivier Harismendy,et al.  Microdroplet-based Pcr enrichment for large-scale targeted sequencing , 2016 .

[33]  S. Elledge,et al.  Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC , 2007, Nature Methods.

[34]  Jean Peccoud,et al.  A syntactic model to design and verify synthetic genetic constructs derived from standard biological parts , 2007, Bioinform..

[35]  Nicolas Jaccard,et al.  Microfluidic approaches for systems and synthetic biology. , 2010, Current opinion in biotechnology.

[36]  E. Andrianantoandro,et al.  Synthetic biology: new engineering rules for an emerging discipline , 2006, Molecular systems biology.

[37]  Tom Ellis,et al.  DNA assembly for synthetic biology: from parts to pathways and beyond. , 2011, Integrative biology : quantitative biosciences from nano to macro.

[38]  Jean Peccoud,et al.  Writing DNA with GenoCAD™ , 2009, Nucleic Acids Res..

[39]  Nathan J Hillson,et al.  j5 DNA assembly design automation. , 2014, Methods in molecular biology.