University of California

Novozymes A/s

The Scripps Research Institute

Robert Blazej

Richard A Mathies

Palani Kumaresan

Chung N Liu

Stephanie H I Yeung

Charles Emrich

Brian M Paegel

Chaoyang Yang

Richard N Cohen

Nicholas Toriello

Nitzan Koppel

This invention provides novel variant cellulolytic enzymes having improved activity and/or stability. In certain embodiments the variant cellulotyic enzymes comprise a glycoside hydrolase with or comprising a substitution at one or more positions corresponding to one or more of residues F64, A226, and/or E246 inCelA enzyme. In certain embodiments the glycoside hydrolase is a variant of a family 9 glycoside hydrolase. In certain embodiments the glycoside hydrolase is a variant of a theme B family 9 glycoside hydrolase.

Cellulase variants

Methods and microfluidic circuitry for inline injection of nucleic acids for capillary electrophoresis analysis are provided. According to various embodiments, microfabricated structures including affinity-based capture matrixes inline with separation channels are provided. The affinity-based capture matrixes provide inline sample plug formation and injection into a capillary electrophoresis channel. Also provided are methods and apparatuses for a microbead-based inline injection system for DNA sequencing.

Inline-injection microdevice and microfabricated integrated DNA analysis system using same

Described are methods and means for enhancing enzyme activity toward insoluble substrates. This is achieved by means of in vitro compartmentalization in which an insoluble microparticle functions both as the enzyme substrate and as a structure for negative selection. Enhanced enzymes expressed from a microparticle-linked polynucleotide library preferentially degrade the microparticle releasing specific gene variants into solution. Gene variants encoding less active enzyme variants remain linked to the microparticle and may be removed through centrifugation, thus enriching the polynucleotide library for more active enzyme variants. These methods may be used to enhance cellulase and ligninase activity toward insoluble cellulosic biomass.

Method of enhancing enzyme activity

Provided are microfluidic designs and methods for rapid generation of monodisperse nanoliter volume droplets of reagent/target (e.g., molecule or cell) mix in emulsion oil. The designs and methods enable high-throughput encapsulation of a single target (e.g., DNA/RNA molecules or cells) in controlled size droplets of reagent mix. According to various embodiments, a microfabricated, 3-valve pump is used to precisely meter the volume of reagent/target mix in each droplet and also to effectively route microparticles such as beads and cells into the device, which are encapsulated within droplets at the intersection of the reagent channel and an oil channel. The pulsatile flow profile of the microfabricated pumps provides active control over droplet generation, thereby enabling droplet formation with oils that are compatible with biological reactions but are otherwise difficult to form emulsions with.

Microfabricated droplet generator for single molecule/cell genetic analysis in engineered monodispersed emulsions

Methods and apparatus for genome analysis are provided. A microfabricated structure including a microfluidic distribution channel is configured to distribute microreactor elements having copies of a sequencing template into a plurality of microfabricated thermal cycling chambers. A microreactor element may include a microcarrier element carrying the multiple copies of the sequencing template. The microcarrier element may comprise a microsphere. An autovalve at an exit port of a thermal cycling chamber, an optical scanner, or a timing arrangement may be used to ensure that only one microsphere will flow into one thermal cycling chamber wherein thermal cycling extension fragments are produced. The extension products are captured, purified, and concentrated in an integrated oligonucleotide gel capture chamber. A microfabricated component separation apparatus is used to analyze the purified extension fragments. The microfabricated structure may be used in a process for performing sequencing and other genetic analysis of DNA or RNA.

Growing community of inventors

San Francisco, CA, United States of America

Robert Blazej