title: A Versatile Microfluidic Device for Automating Synthetic Biology.
abstract: New microbes are being engineered that contain the genetic circuitry, metabolic pathways, and other cellular functions required for a wide range of applications such as producing biofuels, biobased chemicals, and pharmaceuticals. Although currently available tools are useful in improving the synthetic biology process, further improvements in physical automation would help to lower the barrier of entry into this field. We present an innovative microfluidic platform for assembling DNA fragments with 10× lower volumes (compared to that of current microfluidic platforms) and with integrated region-specific temperature control and on-chip transformation. Integration of these steps minimizes the loss of reagents and products compared to that with conventional methods, which require multiple pipetting steps. For assembling DNA fragments, we implemented three commonly used DNA assembly protocols on our microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). We demonstrate the utility of these methods by assembling two combinatorial libraries of 16 plasmids each. Each DNA plasmid is transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. We anticipate that this platform will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids and will help to expedite the overall synthetic biology process.
authors.affiliation: Sandia National Laboratories , 7011 East Avenue, Livermore, California 94550, United States.
authors.forename: Steve C C
authors.initials: SC
authors.lastname: Shih
authors.affiliation: Physical Bioscience Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Garima
authors.initials: G
authors.lastname: Goyal
authors.affiliation: Sandia National Laboratories , 7011 East Avenue, Livermore, California 94550, United States.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Kim
authors.affiliation: Physical Bioscience Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Koutsoubelis
authors.affiliation: Physical Bioscience Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.|Department of Chemical & Biomolecular Engineering, Department of Bioengineering, University of California , Berkeley, California 94720, United States.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Physical Bioscience Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.affiliation: Physical Bioscience Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Hillson
authors.affiliation: Sandia National Laboratories , 7011 East Avenue, Livermore, California 94550, United States.
authors.forename: Anup K
authors.initials: AK
authors.lastname: Singh
mesh_terms: D004247:DNA / Q000737:chemistry / Q000235:genetics; D056656:Lab-On-A-Chip Devices; D058615:Synthetic Biology / Q000295:instrumentation*
chemical_list: D004247:DNA
keywords: DNA assembly; Gibson assembly; Golden Gate assembly; TAR cloning; digital microfluidics; droplet microfluidics; synthetic biology; yeast assembly