Solid phase enzyme kinetics screening in microcolonies
AssigneeKairos Scientific, Inc.
InventorsWilliam J. Coleman, Edward J. Bylina, Douglas C. Youvan
Life of this patent
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Abstract
Improvements in calorimetric assays, surfaces for arraying microcolonies and instrument hardware for screening mutagenized enzymes and proteins in a solid phase format are presented. These improvements permit new enzyme activities to be screened. New filter membrane materials and formats for arraying the microcolonies provide higher throughput, better solvent resistance and ease of handling. Modifications to the instrument heating and illumination systems provide improved temperature control and a more compact, folded light path.
Claims175
A method for imaging and analyzing microcolonies of cells, including the steps of:
- forming in excess of 100 regions, each region including at least one biological cell on a substantially continuous base at an average density of at least about 10 regions per square centimeter, the base comprising a microporous membrane;
- initiating a chemical reaction in such regions that results in an optically detectable signal that changes over time;
- automatically optically monitoring each region over time for changes in the optical signal of portions of such regions;
- and
- indicating which of such portions have a desired change in optical signal.
A method of performing solid-phase directed evolution enzyme screening, including the steps of:
- generating an average density of at least 10 microcolonies of cells per square centimeter on a solid phase, the solid phase comprising a microporous membrane, and wherein a plurality of microcolonies express variants of at least one enzyme;
- contacting the expressed variants with at least one optical signal substrate, each indicative of a desired enzymatic activity;
- and
- automatically detecting changes over time in one or more optical signals generated by one or more optical signal substrates in the microcolonies, wherein such changes indicate desired enzymatic activity of the variants of the enzyme.
A method of performing solid-phase enzyme discovery screening, including the steps of:
- generating a density of at least 10 microcolonies of cells per square centimeter on a solid phase, the solid phase comprising a microporous membrane, and wherein a plurality of microcolonies consist of cells containing members of a recombinant DNA library;
- contacting the microcolonies with at least one optical signal substrate, each indicative of a desired enzymatic activity;
- and
- automatically detecting changes over time in one or more optical signals generated by one or more optical signal substrates in the microcolonies, wherein such changes indicate desired enzymatic activity.
A method of performing solid-phase directed evolution enzyme screening, wherein the solid phase comprises a track-etch membrane.
A method of performing solid phase directed evolution enzyme screening, wherein the solid phase comprises a microporous membrane whose thickness is less than or equal to about 20 micrometers.
A method of performing solid phase directed evolution enzyme screening, wherein the solid phase comprises a microporous membrane wherein the path of the pores through the microporous membrane is non-tortuous.
A method of performing solid phase directed evolution enzyme screening, wherein the solid phase comprises a microporous membrane which is optically transparent.
A method of performing solid phase directed evolution enzyme screening, wherein the solid phase comprises a microporous membrane which is optically translucent.
A method of performing solid phase directed evolution enzyme screening, wherein the solid phase comprises a microporous membrane, and wherein the mass of the microporous membrane is less than 100 times the mass of substrate solution absorbed or adsorbed by the membrane.
A method of performing solid phase directed evolution enzyme screening, wherein the solid phase comprises a microporous membrane, and wherein the mass of the microporous membrane is less than 10 times the mass of substrate solution absorbed or adsorbed by the membrane.
A method of performing solid phase directed evolution enzyme screening, wherein the solid phase comprises a microporous membrane, and wherein the mass of the microporous membrane is less than or equal to the mass of substrate solution absorbed or adsorbed by the membrane.
A method of performing solid-phase enzyme discovery screening, wherein the solid phase comprises a track-etch membrane.
A method of performing solid-phase enzyme discovery screening, wherein the solid phase comprises a microporous membrane whose thickness is less than or equal to about 20 micrometers.
A method of performing solid-phase enzyme discovery screening, wherein the solid phase comprises a microporous membrane in which the path of the pores through the microporous membrane is non-tortuous.
A method of performing solid-phase enzyme discovery screening, wherein the solid phase comprises a microporous membrane which is optically transparent.
A method of performing solid-phase enzyme discovery screening, wherein the solid phase comprises a microporous membrane which is optically translucent.
A method of performing solid-phase enzyme discovery screening, wherein the solid phase comprises a microporous membrane where the mass of the microporous membrane is less than 100 times the mass of substrate solution absorbed or adsorbed by the membrane.
A method of performing solid-phase enzyme discovery screening, wherein the solid phase comprises a microporous membrane where the mass of the microporous membrane is less than 10 times the mass of substrate solution absorbed or adsorbed by the membrane.
A method of performing solid-phase enzyme discovery screening, wherein the solid phase comprises a microporous membrane where the mass of the microporous membrane is less than or equal to the mass of substrate solution absorbed or adsorbed by the membrane.
A method for imaging and analyzing microcolonies of cells, including the steps of:
- forming in excess of 100 regions, each region including at least one biological cell on a substantially continuous base at an average density of at least about 10 regions per square centimeter;
- initiating a chemical reaction in such regions that generates a reaction product and results in an optically detectable signal that changes over time, wherein the signal arises from an interaction between the reaction product and an indicator;
- automatically optically monitoring each region over time for changes in the optical signal of portions of such regions;
- and
- indicating which of such portions have a desired change in optical signal.
A method of performing solid-phase directed evolution enzyme screening, including the steps of:
- generating an average density of at least 10 microcolonies of cells per square centimeter on a solid phase, wherein a plurality of microcolonies express variants of at least one enzyme;
- contacting the expressed variants with at least one optical signal substrate, each indicative of a desired enzymatic activity;
- wherein the optical signal substrate interacts directly with the enzyme being screened, and
- automatically detecting changes over time in one or more optical signals generated by one or more optical signal substrates in the microcolonies, wherein such changes indicate desired enzymatic activity of the variants of the enzyme.
A method of performing solid-phase directed evolution enzyme screening, including the steps of:
- generating an average density of at least 10 microcolonies of cells per square centimeter on a solid phase, wherein a plurality of microcolonies express variants of at least one enzyme;
- contacting the expressed variants with at least one optical signal substrate, each indicative of a desired enzymatic activity;
- and
- automatically detecting changes over time in one or more optical signals generated by one or more optical signal substrates in the microcolonies, wherein the signal arises from an interaction between a product of the enzymatic activity and the optical signal substrate, and wherein such changes indicate desired enzymatic activity of the variants of the enzyme.
A method of performing solid-phase enzyme discovery screening, including the steps of:
- generating a density of at least 10 microcolonies of cells per square centimeter on a solid phase, wherein a plurality of microcolonies consist of cells containing members of a recombinant DNA library;
- contacting the microcolonies with at least one optical signal substrate, each indicative of a desired enzymatic activity;
- and
- automatically detecting changes over time in one or more optical signals generated by one or more optical signal substrates in the microcolonies, wherein the signal arises from an interaction between a product of the enzymatic activity and the optical signal substrate, and wherein such changes indicate desired enzymatic activity.
A method for imaging and analyzing microcolonies of cells, including the steps of:
- forming in excess of 100 regions, each region including at least one biological cell on a substantially continuous base at an average density of at least about 10 regions per square centimeter;
- initiating a chemical reaction in such regions that results in an optically detectable signal that changes over time, wherein at least one of the reactants participating in the chemical reaction has a molecular weight of at least about 1,000 daltons;
- automatically optically monitoring each region over time for changes in the optical signal of portions of such regions;
- and
- indicating which of such portions have a desired change in optical signal.
A method for imaging and analyzing microcolonies of cells, including the steps of:
- forming in excess of 100 regions, each region including at least one biological cell on a substantially continuous base at an average density of at least about 10 regions per square centimeter;
- initiating a chemical reaction in such regions that results in an optically detectable signal that changes over time, wherein at least one of the reactants participating in the chemical reaction has a molecular weight of at least about 10,000 daltons;
- automatically optically monitoring each region over time for changes in the optical signal of portions of such regions;
- and
- indicating which of such portions have a desired change in optical signal.
A method for imaging and analyzing microcolonies of cells, including the steps of:
- forming in excess of 100 regions, each region including at least one biological cell on a substantially continuous base at an average density of at least about 10 regions per square centimeter;
- initiating a chemical reaction in such regions that results in an optically detectable signal that changes over time, wherein at least one of the reactants participating in the chemical reaction has a molecular weight of at least about 100,000 daltons;
- automatically optically monitoring each region over time for changes in the optical signal of portions of such regions;
- and
- indicating which of such portions have a desired change in optical signal.
A method for imaging and analyzing microcolonies of cells, including the steps of:
- forming in excess of 100 regions, each region including at least one biological cell on a substantially continuous base at an average density of at least about 10 regions per square centimeter;
- initiating a chemical reaction in such regions that generates a reaction product and results in an optically detectable signal that changes over time, wherein the signal arises through the action of an indicator enzyme that converts the reaction product into optically detectable product;
- automatically optically monitoring each region over time for changes in the optical signal of portions of such regions;
- and
- indicating which of such portions have a desired change in optical signal.
A method for imaging and analyzing microcolonies of cells, including the steps of:
- forming in excess of 100 regions, each region including at least one biological cell on a substantially continuous base at an average density of at least about 10 regions per square centimeter, wherein region formation includes the step of applying an inoculum to the substantially continuous base;
- initiating a chemical reaction in such regions that results in an optically detectable signal that changes over time;
- automatically optically monitoring each region over time for changes in the optical signal of portions of such regions;
- and
- indicating which of such portions have a desired change in optical signal.
A method for imaging and analyzing microcolonies of cells, including the steps of:
- forming in excess of 100 regions, each region including at least one biological cell on a substantially continuous base at an average density of at least about 10 regions per square centimeter;
- initiating a chemical reaction in such regions that results in an optically detectable signal that changes over time, wherein the initiating comprises the use of a wick;
- automatically optically monitoring each region over time for changes in the optical signal of portions of such regions;
- and
- indicating which of such portions have a desired change in optical signal.
An instrument for imaging and analyzing microcolonies of cells on a target, including:
- a light source for controllably emitting light having a selected set of wavelengths;
- a camera for controllably imaging light received from the target within a selected set of wavelengths;
- a sampling mechanism for controllably selecting samples from the target;
- a processor, coupled to the light source, the camera, and the sampling mechanism, for controlling the wavelengths of light emitted from the light source, the wavelengths of light imaged by the camera, and operation of the sampling mechanism;
- wherein the instrument automatically images regions of the target over time for changes in rate in any optical signal of portions of such regions, and automatically indicates which of such portions have a desired change over time in optical signal, wherein the instrument further comprises a diffuser interposed between the light source and the target for dispersing the emitted light to uniformly illuminate the target.
An instrument for imaging and analyzing microcolonies of cells on a target, including:
- a light source for controllably emitting light having a selected set of wavelengths;
- a camera for controllably imaging light received from the target within a selected set of wavelengths;
- a sampling mechanism for controllably selecting samples from the target;
- a processor, coupled to the light source, the camera, and the sampling mechanism, for controlling the wavelengths of light emitted from the light source, the wavelengths of light imaged by the camera, and operation of the sampling mechanism;
- wherein the instrument automatically images regions of the target over time for changes in rate in any optical signal of portions of such regions, and automatically indicates which of such portions have a desired change over time in optical signal, wherein the instrument further comprises a folded light path between the light source and the camera.
An instrument for imaging and analyzing microcolonies of cells on a target, including:
- a light source for controllably emitting light having a selected set of wavelengths;
- a camera for controllably imaging light received from the target within a selected set of wavelengths;
- a sampling mechanism for controllably selecting samples from the target;
- a processor, coupled to the light source, the camera, and the sampling mechanism, for controlling the wavelengths of light emitted from the light source, the wavelengths of light imaged by the camera, and operation of the sampling mechanism;
- wherein the instrument automatically images regions of the target over time for changes in rate in any optical signal of portions of such regions, and automatically indicates which of such portions have a desired change over time in optical signal, wherein the instrument further comprises a temperature-controlled forced-air heating system for maintaining the temperature of the microcolonies of cells on the target.
A method of performing solid-phase directed evolution enzyme screening, including the steps of:
- generating an average density of at least 10 microcolonies of cells per square centimeter on a solid phase, wherein a plurality of microcolonies express variants of at least one enzyme, wherein the method used to generate the variants is rational design;
- contacting the expressed variants with at least one optical signal substrate, each indicative of a desired enzymatic activity;
- and
- automatically detecting changes over time in one or more optical signals generated by one or more optical signal substrates in the microcolonies, wherein such changes indicate desired enzymatic activity of the variants of the enzyme.
A method of performing solid-phase directed evolution enzyme screening, including the steps of:
- generating an average density of at least 10 microcolonies of cells per square centimeter on a solid phase, wherein a plurality of microcolonies express variants of at least one enzyme;
- wherein the method used to generate the variants is at least one selected from the group consisting of error-prone PCR, chemical mutagenesis, cassette/cartridge mutagenesis, mutator strain induced mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, DNA shuffling, and sequential random mutagenesis,
- contacting the expressed variants with at least one optical signal substrate, each indicative of a desired enzymatic activity;
- and
- automatically detecting changes over time in one or more optical signals generated by one or more optical signal substrates in the microcolonies, wherein such changes indicate desired enzymatic activity of the variants of the enzyme.
Dependencies are inferred from the claim text.