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.

Figure 1.

Overview of DMF cartridge design. Top: dimensions and general layout of the cartridge, with annotated reservoirs names. Bottom (magnified): annotated schematics of a section of the microfluidics cartridge layout. Different regions corresponding to different functions are highlighted. Dilution buffer reservoir contains the reagent used to perform dilutions to reach single molecule DNA concentrations/ PCR Mix. Other reservoirs are used to hold master mixes that contain the appropriate pairs of primers. During each phase of the DMF program the storage area is continually loaded with the droplets required for the following stages of the run to speed up the protocols. The dilution zone is used to perform the serial dilutions for POP assembly, M-CAD and smPCR based in vitro cloning. The PCR lane consist of three temperatures zones at 62°C, 72°C and 95°C. Temperature ramping is accomplished by DMF-based droplet shuttling between temperature zones.

Figure 2.

DNA Synthesis using DMF. (A) POP assembly schematics. A droplet containing template DNA (gray) is combined with assembly droplet 1 (AD1) that contains the primers and assembly mix to form a reaction droplet (thermo-cycled, in gray) in which assembly product 1 (AP 1) is generated. The AP1 containing droplet is then combined with assembly droplet 2 (AD2) that contains the primers and assembly mix to form a new reaction droplet (thermo-cycled, in gray) in which assembly product 2 (AP2) is generated. The process is iterated (with AD3, AD4, etc.) until the full length molecule (AP4) is constructed. (B) Schematic representation of the RBS DNA library, its transformation into yeast and its gene expression measurement in 96-well format using a plate reader. (C) Gene expression measurements of POP-generated 5′UTR library showing a 10-fold variation in gene expression across library variants. (D) Overview of M-CAD, a method that receives a set of DNA molecules as input and copies segments from them to create variants according to specification in a digital microfluidic device. A. M-CAD enables the implementation of the basic text editing operations on DNA molecules such as Cut, Copy, Paste, Cut and Paste, and Copy and Paste. B. The text editing operations required for constructing a DNA library from a set of DNA inputs (top of the tree) are translated into a tree in which vertices are DNA molecules and edges are the editing operations (cut, paste, etc.) for generating the vertices. Purple represents DNA that was originally present on the input DNA molecules and red represents synthetic DNA added during the editing process (from PCR primers for example). Vertices V1–V10 are the final 10 variant DNA molecules in this example of –CAD construction C. various input DNA molecules and primers are loaded onto the device and droplets generated from them (colored droplets) are routed on cartridge to assemble the target DNA molecules in a pre-defined manner (as determined in B).

Figure 3.

Scheme of cell free cloning using DMF. (A) The full length construct generated by POP assembly is subjected to DMF-based in vitro cloning using smPCR. A POP assembly product is iteratively diluted 2-fold using the following DMF operations: (i) merge (with diluent), (ii) split, (iii) trash (one half) and (iv) recycling of the second half back into the serial dilution. Once diluted, droplets are calculated to contain an average of one target DNA molecule per droplet and the single molecule droplets are programmed to travel to the PCR zone. In the PCR zone, single DNA molecule PCR droplets are amplified by PCR via their travel between the temperature zones. (B) Zoomed in view on the randomized 5′UTR region of a sequencing reaction from the DMF smPCR reactions. The sequencing chromatograms validate that single POP generated molecules were amplified on chip since the randomized 5′UTR sequence also functions as a barcode that verifies clonality (lanes 1–3). In contrast, negative control smPCR experiments (controls with many template molecules) show a clear pattern of variability in the 5′UTR/barcode region (center of chromatogram, lanes 4–8).