Research Tools

Most Recent Inventions

Platform for High-Throughput Analysis of Microbial Interactions

UW–Madison researchers have developed a research tool for large-scale mapping of interactions in microbiomes. Their method employs gene sequencing in a microfluidic system to increase throughput by several orders of magnitude (1,000-10,000 times).

Specifically, the researchers mixed groups of several species of bacteria in culture. They encapsulated cells into millions of picoliter droplets dispersed in an oil phase. The droplets were incubated to allow the microbes to interact, assemble into a community and perform functional activities. After incubation, the composition of the community within the droplet was analyzed using fluorescence microscopy or next-generation DNA sequencing.

The presence or absence of microbes in a drop can be indicative of different species preferentially interacting with other species in the bulk culture or droplet, and can be used to reconstruct the microbiome’s ecological network.

Nanocapsule Delivery System for Ribonucleoproteins

UW–Madison researchers have engineered a biodegradable GSH-responsive nanocoating surrounding the sgRNA/Cas9 RNP complex for efficient delivery into cells. The RNP nanocapsule is a polymeric network synthesized from a mixture of (meth)acrylate monomers, acrylate crosslinkers and acrylate polyethylene glycol (PEG), built around the RNP cargo. The interactions between the RNP and the monomers include electrostatic and hydrophobic interactions and hydrogen bonding. Nanocapsule formation is completed by free radical polymerization.

Each component of this nanocapsule is essential for cellular delivery. Inclusion of the imidazole-containing monomer provided a mechanism for endosomal/lysosomal escape. Without this, the sgRNA/Cas9 RNP would be destroyed before exerting its effects. A crosslinker containing a disulfide (–S-S–) bond produced a covalently linked, yet biodegradable, shell around the cargo. And finally, to reduce potential recognition by the immune system and increase the circulation half-life, a PEG outer shell was introduced. The PEG outer shell also provides a chemical handle for attaching fluorescent dyes or targeting ligands onto the RNP nanocapsule.

S1mplex: A New Tool for Precision Gene Editing

UW–Madison researchers have developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to ‘sharpen the scalpels’ used in genome surgery. In the new approach, CRISPR-Cas9 RNPs are complexed with a nucleic acid donor template, as well as other biotinylated molecules (e.g., quantum dots).

In human pluripotent stem cells, tailored S1mplexes increased the ratio of precisely edited to imprecisely edited alleles up to 18-fold higher than standard gene editing methods, and enriched cell populations containing multiplexed precise edits up to 42-fold.

These advances with versatile, preassembled reagents could greatly reduce the time and cost of in vitro/ex vivo gene editing applications in precision medicine and drug discovery, and aid in the development of increased and multiple dosing regimens for somatic gene editing in vivo.

Creating ‘Designer’ Yeast Hybrids for Brewing and More

UW–Madison researchers have developed HyPr, a simple and efficient method for generating synthetic Saccharomyces hybrids without sporulation or modification of the nuclear genome.

Specifically, using the new method, induction of HO endonuclease expression by a promoter in two diploid cultures, followed by co-culture and subsequent double-drug selection, will produce hybrids at a rate approaching 1 out of 1,000 cells plated. Plasmids can then be easily cured or spontaneously lost to produce strains without genome modifications.

The resulting strains can be rapidly screened for plasmid loss, opening an efficient route towards meeting the Generally Recognized as Safe (GRAS) standards of the U.S. Department of Agriculture and FDA.

Multicolor Reporter Cells for Detecting and Quantifying HIV-1

UW–Madison researchers have developed highly sensitive and specific reporter cell lines suitable for automated detection of HIV in a microfluidic platform. The multicolor fluorescence-based cell readouts respond robustly to HIV-1 infections, and are useful for tracking the spread of HIV-1 infection and ideal for implementation in an automated Q-VOA assay.

The reporter cells are based on coupling fluorescent markers turned ‘on’ in response to HIV infection to markers turned ‘off’ by the virus. Calculating net ‘on/off’ ratios over time, relative to standards, allows for high sensitivity and favorable signal-to-noise. The ability to amplify response signals with minimal background and without the need for chemical substrates represents a significant improvement over existing green fluorescent protein (GFP) or chemiluminescence-based single reporter lines.

Most Recent Patents

Designing Programmable Inducible Promoters for Biosensor Applications

UW–Madison researchers have developed a method for de novo design of synthetic inducible promoters for transcription factors and other DNA binding proteins such as aTFs with tunable dynamic range behavior and compatibility with virtually any host organism.

The method can include selecting inducible promoters, for example, by converting a constitutive promoter of an organism into an inducible promoter by introducing binding sites near the RNA polymerase binding site. By controlling the access of a transcription factor and the RNA polymerase to the promoter, the dynamic range of the system can be controlled.

Circulating Tumor Cell Assay Using Simple Blood Draw

The researchers now have developed the first fully integrated CTC assay that could eliminate the need for painful biopsies. The device leverages verIFAST technology to capture, purify and molecularly analyze CTCs from a single blood sample.

The sample is deposited in a first well and then magnetically drawn through a second well containing an isolation buffer like oil or wax. The increasingly purified cells are drawn into a final well for extraction or further treatment.

The device includes new technical features and is combined with downstream techniques for staining rare cells like CTCs.

Improved Methods and Materials for Transforming Plant Cells

UW-Madison researchers have developed a method of using a DNA-launching platform to introduce viral RNA into a host cell that has been engineered to support viral replication and expression. The platform encodes a modified viral RNA molecule located downstream of a DNA-dependent RNA polymerase promoter. After the platform is introduced into a host cell, it effectively “launches” the RNA molecule into the cell, where the RNA is replicated and expressed.