Synthetic biology aims to improve human health and the environment by repurposing biological enzymes for use in practical applications. However, natural enzymes often function with suboptimal activity when engineered into biological pathways or challenged to recognize unnatural substrates. Overcoming this problem requires efficient directed evolution methods for discovering new enzyme variants that function with a desired activity. Here, we describe the construction, validation, and application of a fluorescence-activated droplet sorting (FADS) instrument that was established to evolve enzymes for synthesizing and modifying artificial genetic polymers (XNAs). The microfluidic system enables droplet sorting at ~2-3 kHz using fluorescent sensors that are responsive to enzymatic activity. The ability to evolve nucleic acid enzymes with customized properties will uniquely drive emerging applications in synthetic biology, biotechnology, and healthcare.

Engineering polymerases to synthesize artificial genetic polymers with unique backbone structures is limited by a general lack of understanding about the structural determinants that govern substrate specificity. Here, we report a high-throughput microfluidic-based approach for mapping sequence–function relationships that combines droplet-based optical polymerase sorting with deep mutational scanning. We applied this strategy to map the finger subdomain of a replicative DNA polymerase isolated from Thermococcus kodakarensis (Kod). The enrichment profile provides an unbiased view of the ability of each mutant to synthesize threose nucleic acid, which was used as a model non-natural genetic polymer. From a single round of sorting, we discovered two cases of positive epistasis and demonstrate the near inversion of substrate specificity from a double mutant variant. This effort indicates that polymerase specificity may be governed by a small number of highly specific residues that can be elucidated by deep mutational scanning without the need for iterative rounds of directed evolution.

Most DNA polymerase libraries sample unknown portions of mutational space and are constrained by the limitations of random mutagenesis. Here we describe a programmed allelic mutagenesis (PAM) strategy to comprehensively evaluate all possible single-point mutations in the entire catalytic domain of a replicative DNA polymerase. By applying the PAM strategy with ultrafast high-throughput screening, we show how DNA polymerases can be mapped for allelic mutations that exhibit enhanced activity for unnatural nucleic acid substrates. We suggest that comprehensive missense mutational scans may aid the discovery of specificity determining residues that are necessary for reprogramming the biological functions of natural DNA polymerases.

Organic fluorescent dyes are widely used for the visualization of bound antibody in a variety of immunofluorescence assays. However, the detection equipment is often expensive, fragile and hard to deploy widely. Quantum dots (Qdot®) are nanocrystals made of semiconductor materials that emit light at different wavelengths according to the size of the crystal, with increased brightness and stability. Here we have evaluated a small benchtop ‘personal’ optical imager (ArrayCAM™) developed for quantification of protein arrays probed by Qdot -based indirect immunofluorescence. The aim was to determine if the Qdot imager system provides equivalent data to the conventional organic dye-labelled antibody/laser scanner system. To do this, duplicate proteome microarrays of Vaccinia virus, Brucella melitensis and Plasmodium falciparum were probed with identical samples of immune sera, and IgG, IgA and IgM profiles visualized using biotinylated secondary antibodies followed by a tertiary reagent of streptavidin coupled to either P3 (an organic cyanine dye typically used for microarrays) or Q800 (Qdot). The data show excellent correlation for all samples tested (R>0.8) with no significant change of antibody reactivity profiles. We conclude that Qdot detection provides data equivalent to that obtained using conventional organic dye detection. The portable imager offers an economical, more robust and deployable alternative to conventional laser array scanners.

Immunotherapy This image depicts cell‐mimicking artificial antigen‐presenting cells (aAPCs, pink) and biological T cells (blue). In article 2203163 by Anshu Agrawal, Abraham P. Lee, and co‐workers, aAPCs are produced using a microfluidic device, which enables facile and stable double emulsion droplet generation. By recapitulating the properties of a cell, namely, size, fluidity, and surface proteins, aAPCs are able to engage with T cells, forming immune synapses. Multiple ligands on aAPCs can engage with the receptors on T cells due to the high surface fluidity of the lipid bilayer. The size of the aAPC (≈20 μm diameter) also contributes to an adequate force that is essential for T cell activation. Experimental results suggest that T cells can tightly bind with the aAPCs and maximize their contact area. After interacting with aAPCs, T cells are activated and subsequently proliferate in large numbers. The background shows a wide spectrum of aAPCs, T cells, and most importantly, aAPC‐T cell pairs joint by immune synapses, as is observed in this research.