Quantitative Tracking of Combinatorially Engineered Populations with Multiplexed Binary Assemblies.

Advances in synthetic biology and genomics have enabled full-scale genome engineering efforts on laboratory time scales. However, the absence of sufficient approaches for mapping engineered genomes at system-wide scales onto performance has limited the adoption of more sophisticated algorithms for engineering complex biological systems. Here we report on the development and application of a robust approach to quantitatively map combinatorially engineered populations at scales up to several dozen target sites.

Genome scale engineering techniques for metabolic engineering.

Metabolic engineering has expanded from a focus on designs requiring a small number of genetic modifications to increasingly complex designs driven by advances in genome-scale engineering technologies. Metabolic engineering has been generally defined by the use of iterative cycles of rational genome modifications, strain analysis and characterization, and a synthesis step that fuels additional hypothesis generation. This cycle mirrors the Design-Build-Test-Learn cycle followed throughout various engineering fields that has recently become a defining aspect of synthetic biology.

Multiplexed tracking of combinatorial genomic mutations in engineered cell populations.

Multiplexed genome engineering approaches can be used to generate targeted genetic diversity in cell populations on laboratory timescales, but methods to track mutations and link them to phenotypes have been lacking. We present an approach for tracking combinatorial engineered libraries (TRACE) through the simultaneous mapping of millions of combinatorially engineered genomes at single-cell resolution. Distal genomic sites are assembled into individual DNA constructs that are compatible with next-generation sequencing strategies.

Codon compression algorithms for saturation mutagenesis.

Saturation mutagenesis is employed in protein engineering and genome-editing efforts to generate libraries that span amino acid design space. Traditionally, this is accomplished by using degenerate/compressed codons such as NNK (N = A/C/G/T, K = G/T), which covers all amino acids and one stop codon. These solutions suffer from two types of redundancy: (a) different codons for the same amino acid lead to bias, and (b) wild type amino acid is included within the library.

Active control of nanolitre droplet contents with convective concentration gradients across permeable walls.

Nanolitre droplets in microfluidic devices can be used to perform thousands of independent chemical and biological experiments while minimizing reagents, cost and time. However, the absence of simple and versatile methods capable of controlling the contents of these nanolitre chemical systems limits their scientific potential. To address this, we have developed a method that is simple to fabricate and can continuously control nanolitre chemical systems by integrating a time-resolved convective flow signal across a permeable membrane wall.

A Venturi microregulator array module for distributed pressure control.

Pressure-driven flow control systems are a critical component in many microfluidic devices. Compartmentalization of this functionality into a stand-alone module possessing a simple interface would allow reduction of the number of pneumatic interconnects required for fluidic control. Ideally, such a module would also be sufficiently compact for implementation in portable platforms.

Selective arraying of complex particle patterns.

Procedures requiring precise and accurate positioning of particles and cells have impacted a broad range of research interests including molecular detection, self-assembly and tissue and cell engineering. These fields would be greatly aided by more advanced, yet straightforward, micro-object positioning methods that are precise, scalable, responsive and flexible. We have developed an arrayed, multilayer surface patterned microfluidic device which uses laminar convective flow to actively position particles into any desired, two-dimensional, predesigned pattern.

Acoustically driven programmable liquid motion using resonance cavities.

Performance and utility of microfluidic systems are often overshadowed by the difficulties and costs associated with operation and control. As a step toward the development of a more efficient platform for microfluidic control, we present a distributed pressure generation scheme whereby independently tunable pressure sources can be simultaneously controlled by using a single acoustic source. We demonstrate how this scheme can be used to perform precise droplet positioning as well as merging, splitting, and sorting within open microfluidic networks.

Transverse imaging and simulation of dsDNA electrophoresis in microfabricated glass channels.

We have observed the non-uniform distribution of DNA molecules during PAGE in a microfabricated system. Confocal laser scanning microscopy was used to visualize fluorescently labeled DNA during electrophoretic migration. The distribution of double-stranded DNA larger than 100 bp is observed to transition from a center-biased motion on the transverse plane 1 cm downstream from injection to an edge-biased motion 2 cm downstream.