Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

Abstract

Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.

Droplet microfluidics for synthetic biology

“…In contrast, digital droplet formation methods, while offering lower-throughput, provide ondemand droplet manipulation and control. 10,11 Digital microfluidics frequently use electrowetting-on-dielectric (EWOD) in which a voltage applied to an electrode pad (lowering the contact angle of the droplet on the hydrophobic surface and macroscopically converting the surface to hydrophilic) provides a droplet-driving force. 12,13 Droplets can also be manipulated using dielectrophoretic (DEP) forces using an array of electrodes.…”

“…The DMF platform used by Yehezkel et al was successfully applied to the assembly of doublestranded DNA, starting with 160 bp single-stranded oligos that are taken through a series of additions in 300 nL droplets. 10 Beyond oligo assembly, such a system, if scaled using a solid-phase substrate, could potentially implement the consecutive rounds of reaction and washing steps required for standard phosphoramidite-based DNA synthesis. 27 Ongoing efforts are needed to further increase the number of sample preparation steps that can be performed on-chip to translate these technologies into fully integrated systems.…”

“…2b). 10 The oligonucleotides were assembled using a programmable order polymerization (POP) assembly process in which DNA fragments are iteratively added to elongate an initial DNA template. This group adapted the PCBbased DMF platform developed by Advanced Liquid Logic (later acquired by Illumina, Inc.) to perform these operations.…”

“…The chip was also applied to the combinatorial assembly of a variant library of the Azurine gene. 10 This system successfully completed 24 assembly reactions requiring 50-fold less reagents and 10 times less time than conventional manual library preparation. The same device could also be used for performing single-molecule PCR to allow for cell-free DNA cloning.…”

An end-to-end solution for automation of biological protocols

“…However, in recent years there has been an increased interest in automation and many innovative and low-cost solutions have been proposed and launched. 40 , purification 41 , cloning 42 and transformation/transfection 43,44 . There have been many platforms developed which allow users to conduct these experiments in an integrated manner 45 .…”