Micro-screening apparatus, process, and products
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsJennifer R. Cochran, Thomas M. Baer, Bob Chen, Spencer Caleb Alford, Arvind Kannan, Sungwon Lim, Ivan Dimov
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Abstract
Microcavity arrays and methods for quantitative biochemical and biophysical analysis of populations of biological variants. Examples include high-throughput analysis of cells and protein products use a range of fluorescent assays, including binding-affinity measurement and time-resolved enzyme assays. Laser-based extraction of microcavity contents.
Claims19
A method for screening a library of cells having a plurality of genotypes for a cell having a phenotype of interest for producing a molecule of interest, the method comprising
- (a) loading a microcavity array with the library of cells;
- (b) incubating the array under conditions that allow for production of the molecule of interest;
- (c) imaging the array to identify a cavity comprising cells having the phenotype of interest;
- and
- (d) extracting the contents of the cavity comprising cells having the phenotype of interest by directing electromagnetic radiation from a pulsed diode laser that delivers electromagnetic radiation in 2-20 pulses having pulse length of 1-10milliseconds with a pulse separation of 10 to 100 millisecond at a radiation absorbing material associated with the cavity.
A method for engineering a property of interest in a fluorescent protein, wherein the property of interest comprises at least one of an emission spectra, an emission intensity, a stokes shift, and an absorption spectra, the method comprising creating a library of cells producing a mutant form of a fluorescent protein, and screening the library for cells producing the mutant form having the property of interest, wherein the screening method comprises:
- (a) loading a microcavity array with the library of cells;
- (b) incubating the array under conditions that allow for production of the mutant form;
- (c) imaging the array to identify a cavity comprising cells producing the mutant form;
- and
- (d) extracting the contents of the cavity comprising cells producing the mutant form by directing electromagnetic radiation from a pulsed diode laser that delivers electromagnetic radiation in 2-20 pulses having pulse length of 1-10milliseconds with a pulse separation of 10 to 100 millisecond at a radiation absorbing material associated with the cavity.
A method for measuring enzyme kinetics for a member of a protein enzyme library produced by a library of cells having a plurality of genotypes for producing mutant forms of a protein enzyme;
- the method comprising
- (a) loading a microcavity array with the library of cells;
- (b) incubating the array in the presence of a substrate for the protein enzyme under conditions that allow for production of the enzyme of interest;
- (c) imaging the array at selected intervals;
- (d) measuring difference in enzyme activity for one or more cavities of the array;
- (e) extracting the contents of a cavity comprising cells having the phenotype of interest by directing electromagnetic radiation from a pulsed diode laser that delivers electromagnetic radiation in 2-20 pulses having pulse length of 1-10milliseconds with a pulse separation of 10 to 100 millisecond at a radiation absorbing material associated with the cavity.
Dependencies are inferred from the claim text.