Microfluidic devices are valuable for a variety of biotechnology applications, such as synthesizing biochemical libraries, screening enzymes, and analyzing single cells. However, normally, the devices are controlled using specialized pumps, which require expert knowledge to operate. Here, we demonstrate operation of poly(dimethylsiloxane) devices without pumps. We build a scaffold that holds the device and reagents to be infused in a format that can be inserted into a 50 mL falcon tube and spun in a common lab centrifuge. By controlling the device design and centrifuge spin speed, we infuse the reagents at controlled flow rates. We demonstrate the encapsulation and culture of clonal colonies of red and green Escherichia coli in droplets seeded from single cells.

Microfluidic devices can form double emulsions with uniform properties, but require cumbersome fabrication steps to pattern their wettability. We demonstrate spatially-controlled plasma oxidation to create wettability patterns for forming double emulsions. Our method performs comparably to chemical techniques but is simpler, more reliable, and scalable to patterning large arrays of drop makers.

Genetic heterogeneity is an important feature of many biological systems, but introduces technical challenges to their characterization. Even with the best modern instruments, only a small fraction of DNA molecules present in a sample can be read, and they are recovered in the form of short, hundred-base reads. In this paper, we introduce 3dPCR, a method to sort DNA molecules with sequence specificity. 3dPCR allows heterogeneous populations of DNA to be sorted to recover long targets for deep sequencing. It is valuable whenever a target sequence is rare in a mixed population, such as for characterizing mutations in heterogeneous cancer cell populations or identifying cells containing a specific genetic sequence or infected with a target virus.

Currently, the identification of new genes drastically outpaces current experimental methods for determining their enzymatic function. This disparity necessitates the development of high-throughput techniques that operate with the same scalability as modern gene synthesis and sequencing technologies. In this paper, we demonstrate the versatility of the recently reported DNA-Linked Enzyme-Coupled Assay (DLEnCA) and its ability to support high-throughput data acquisition through next-generation sequencing (NGS). Utilizing methyltransferases, we highlight DLEnCA's ability to rapidly profile an enzyme's substrate specificity, determine relative enzyme kinetics, detect biosynthetic formation of a target molecule, and its potential to benefit from the scales and standardization afforded by NGS. This improved methodology minimizes the effort in acquiring biosynthetic knowledge by tying biochemical techniques to the rapidly evolving abilities in sequencing and synthesizing DNA.

Droplet microfluidics is valuable for applications in chemistry and biology, but generates massive numbers of droplets that must be analyzed and sorted. Here, we describe a simple approach to bulk double emulsify microfluidic emulsions for analysis and sorting with commercial flow cytometers. We illustrate the method by using it to identify droplets based on nucleic acid content. Though simple, our method provides a general approach for analyzing and sorting microfluidic droplets without custom microfluidic double emulsifiers or sorters.

Our ability to engineer organisms with new biosynthetic pathways and genetic circuits is limited by the availability of protein characterization data and the cost of synthetic DNA. With new tools for reading and writing DNA, there are opportunities for scalable assays that more efficiently and cost effectively mine for biochemical protein characteristics. To that end, we have developed the Multiplex Library Synthesis and Expression Correction (MuLSEC) method for rapid assembly, error correction, and expression characterization of many genes as a pooled library. This methodology enables gene synthesis from microarray-synthesized oligonucleotide pools with a one-pot technique, eliminating the need for robotic liquid handling. Post assembly, the gene library is subjected to an ampicillin based quality control selection, which serves as both an error correction step and a selection for proteins that are properly expressed and folded in E. coli. Next generation sequencing of post selection DNA enables quantitative analysis of gene expression characteristics. We demonstrate the feasibility of this approach by building and testing over 90 genes for empirical evidence of soluble expression. This technique reduces the problem of part characterization to multiplex oligonucleotide synthesis and deep sequencing, two technologies under extensive development with projected cost reduction.