title: DNA-library assembly programmed by on-demand nano-liter droplets from a custom microfluidic chip.
abstract: Nanoscale synthetic biology can benefit from programmable nanoliter-scale processing of DNA in microfluidic chips if they are interfaced effectively to biochemical arrays such as microwell plates. Whereas active microvalve chips require complex fabrication and operation, we show here how a passive and readily fabricated microchip can be employed for customizable nanoliter scale pipetting and reaction control involving DNA. This recently developed passive microfluidic device, supporting nanoliter scale combinatorial droplet generation and mixing, is here used to generate a DNA test library with one member per droplet exported to addressed locations on microwell plates. Standard DNA assembly techniques, such as Gibson assembly, compatible with isothermal on-chip operation, are employed and checked using off-chip PCR and assembly PCR. The control of output droplet sequences and mixing performance was verified using dyes and fluorescently labeled DNA solutions, both on-chip and in external capillary channels. Gel electrophoresis of products and DNA sequencing were employed to further verify controlled combination and functional enzymatic assembly. The scalability of the results to larger DNA libraries is also addressed by combinatorial input expansion using sequential injection plugs from a multiwell plate. Hence, the paper establishes a proof of principle of the production of functional combinatorial mixtures at the nanoliter scale for one sequence per well DNA libraries.
authors.affiliation: Faculty of Chemistry and Biochemistry, Microsystems Chemistry and BioIT (BioMIP), Ruhr-University Bochum , 44780 Bochum, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Tangen
authors.affiliation: Faculty of Chemistry and Biochemistry, Microsystems Chemistry and BioIT (BioMIP), Ruhr-University Bochum , 44780 Bochum, Germany.
authors.forename: Gabriel Antonio S
authors.initials: GA
authors.lastname: Minero
authors.affiliation: Faculty of Chemistry and Biochemistry, Microsystems Chemistry and BioIT (BioMIP), Ruhr-University Bochum , 44780 Bochum, Germany.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Sharma
authors.affiliation: Faculty of Chemistry and Biochemistry, Microsystems Chemistry and BioIT (BioMIP), Ruhr-University Bochum , 44780 Bochum, Germany.
authors.forename: Patrick F
authors.initials: PF
authors.lastname: Wagler
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science , Rehovot, Israel.
authors.forename: Rafael
authors.initials: R
authors.lastname: Cohen
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science , Rehovot, Israel.
authors.forename: Ofir
authors.initials: O
authors.lastname: Raz
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science , Rehovot, Israel.
authors.forename: Tzipy
authors.initials: T
authors.lastname: Marx
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science , Rehovot, Israel.
authors.forename: Tuval
authors.initials: T
authors.lastname: Ben-Yehezkel
authors.affiliation: Faculty of Chemistry and Biochemistry, Microsystems Chemistry and BioIT (BioMIP), Ruhr-University Bochum , 44780 Bochum, Germany.
authors.forename: John S
authors.initials: JS
authors.lastname: McCaskill