Journal: Nature communications

Article Title: A general strategy for expanding polymerase function by droplet microfluidics.

doi: 10.1038/ncomms11235

Figure Lengend Snippet: Figure 1 | Droplet-based optical polymerase sorting. (a) We have developed a fluorescent reporter system that produces an optical signal when a primer– template complex is extended to full-length product. The reporter consists of a primer–template complex (pink and green) containing a downstream fluorophore that is quenched when a DNA-quencher (black) anneals to the unextended region. (b) The assay was designed with a metastable probe to allow dissociation at elevated temperatures, where thermophilic polymerases function with optimal activity. Red arrow marks the maximium fluorescence observed in the absence of the quencher probe. (c) Flourophore (F)/quencher (Q) pairs were screened to identify a dye pair with the maximum signal-to-noise ratio. (d) Primer-extension analysis by denaturing PAGE (top) and fluorescence (bottom) for 9n and 9n-GLK polymerases using dNTP and NTP substrates. Negative control: no NTPs. Positive control: dNTPs or no DNA-quencher probe. (e) Single-emulsion droplets containing a functional 9n-GLK polymerase that extends a primer–template complex with RNA (top) and non-functional (bottom) wild-type 9n polymerase. The panel shows a cartoon depiction of the droplet, a bright- field micrograph of encapsulated E. coli (arrow), a fluorescence micrograph of the same field of view and an overlay of the two images. Scale bars, 10mm. (f) Flow cytometry analysis of 9n and 9n-GLK polymerases following NTP extension in water-in-oil-in-water (w/o/w) droplets.

Article Snippet: Electrocompetent E. coli cells (50ml, b-10 E. coli cells, NEB) were transformed with 5 ml of purified DNA by applying one electric pulse of 1.80 kV (using an E. coli Pulser cuvette, 0.1 cm electrode; Bio-Rad MicroPulser).

Techniques: Activity Assay, Negative Control, Positive Control, Emulsion, Functional Assay, Flow Cytometry

Journal: Nature communications

Article Title: A general strategy for expanding polymerase function by droplet microfluidics.

doi: 10.1038/ncomms11235

Figure Lengend Snippet: Figure 2 | Model selection of an engineered polymerase with RNA synthesis activity. (a) Overview of the microfluidic polymerase enrichment strategy. A pool of polymerase genes containing functional (green) and non-functional (blue) members are expressed in E. coli and encapsulated in w/o droplets generated in a microfluidics device. Polymerases are liberated from their bacteria by heat lysis and incubated at 55 C to allow for primer extension. Using a second microfluidics device, droplets are emulsified into a bulk aqueous phase to generate water-in-oil-in-water compartments (w/o/w). Fluorescent w/o/ ws are FACS sorted and the vectors encoding functional polymerases are recovered. (b) Vector design. The 9n-GLK vector was engineered to contain a unique NotI restriction site. Control digestion showing that NotI only cuts PCR-amplified DNA from the 9n-GLK vector. (c) Following a complete cycle of selection and amplification (see Supplementary Fig. 1) PCR-amplified DNA was digested with NotI to measure the enrichment of 9n-GLK from libraries that were doped at levels of 1:100, 1:1,000 and 1:10,000 (9n-GLK to 9n). NotI digestion of the PCR-amplified DNA reveals an enrichment of B1,200-fold per round of microfluidics selection.

Article Snippet: Electrocompetent E. coli cells (50ml, b-10 E. coli cells, NEB) were transformed with 5 ml of purified DNA by applying one electric pulse of 1.80 kV (using an E. coli Pulser cuvette, 0.1 cm electrode; Bio-Rad MicroPulser).

Techniques: Selection, Activity Assay, Functional Assay, Generated, Bacteria, Lysis, Incubation, Plasmid Preparation, Control