Systems and methods for identification of optimized protein production and kits therefor
Inventors
McEwen, Jason M. • Lionberger, Troy A. • Sackmann, Eric K. • Kurz, Volker L. S. • Mobilia, Kellen C.
Assignees
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Publication Number
US-12385038-B2
Publication Date
2025-08-12
Expiration Date
Abstract
Systems, methods, and kits therefor, enabling rapid protein evolution are described herein. A system useful in the methods described herein include a DNA synthesis component; a microfluidic system including a microfluidic device having a microfluidic channel and sequestration pens; and a computing component, which is configured to analyze assay results and, based upon the analysis, design improved DNA sequences for iterative protein evolution. The microfluidic system is configured to permit correlation of DNA sequence on a bead to its location within the microfluidic device, permit cell free protein expression of a DNA sequence captured to a bead, and to permit assay of the protein so produced.
Core Innovation
The invention provides a process for evolving a protein using a microfluidic device that includes a housing with a base and a microfluidic structure. The base and the microfluidic structure define a region for holding a first liquid medium with micro-objects suspended therein, and the microfluidic structure provides a flow path and a plurality of chambers fluidically interconnected with the region. A first library of nucleic acid sequences is disposed so that each nucleic acid sequence contains one or more variations from a nucleic acid sequence encoding a protein sequence of interest.
Phenotypic reporter functionality is introduced into the microfluidic device in proximity to each nucleic acid sequence of the first library. The phenotypic reporter includes a solution phase reagent and/or a plurality of micro-objects and is configured to provide a phenotypic readout from the protein sequence of interest. A reagent mixture is also introduced and disposed in proximity to each nucleic acid sequence to express a corresponding protein sequence from each nucleic acid sequence, followed by detecting the phenotypic readout from a region proximal to one or more nucleic acid sequences.
Individual nucleic acid sequences associated with desired phenotypic readouts are identified by determining which proximal regions correspond to a desired phenotypic readout. The nucleotide sequence of the identified nucleic acid sequences of the first library is then determined. The claim coverage further supports microfluidic sequestration, electrode activation, alternative reporter formats, and machine-learning correlation of nucleic acid sequences and phenotypic readouts.
Claims Coverage
The claim coverage centers on a microfluidic protein evolution process with 8 inventive features, together with dependent refinements for microfluidic sequestration, electrode activation, reporter formats, ordering of introduction steps, and machine-learning correlation.
Protein evolving process in a microfluidic device
Disposing a first library of nucleic acid sequences within a microfluidic device having a housing with a base and a microfluidic structure, wherein the base and the microfluidic structure define a region for holding a first liquid medium and micro-objects suspended therein, and wherein the microfluidic structure comprises a flow path and a plurality of chambers fluidically interconnected with the region, and wherein each nucleic acid sequence comprises one or more variations from a nucleic acid sequence encoding a protein sequence of interest.
Proximal phenotypic reporter and expression in microfluidics
Introducing a phenotypic reporter into the microfluidic device in proximity to each nucleic acid sequence, where the phenotypic reporter comprises a solution phase reagent and/or a plurality of micro-objects and is configured to provide a phenotypic readout from the protein sequence of interest; introducing a reagent mixture in proximity to each nucleic acid sequence so that a corresponding protein sequence is expressed from each nucleic acid sequence.
Proximal phenotypic detection, sequence identification, and nucleotide determination
Detecting the phenotypic readout from a region proximal to one or more nucleic acid sequences; identifying individual nucleic acid sequences having a corresponding proximal region with a desired phenotypic readout; and determining the nucleotide sequence of the identified nucleic acid sequences.
Photoconductive or phototransistor DEP electrode activation substrate in microfluidics
Using a microfluidic device with a dielectrophoresis configuration having first and second electrodes in different walls of the housing, and using an electrode activation substrate made of a photoconductive material, semiconductor integrated circuits, or phototransistors.
Sequestration pen structure for biological micro-objects
Using a sequestration pen process including an enclosure with a single opening to a flow path to hold a biological micro-object suspended in a second liquid medium within an interior space.
Sequencing of reporter and reagent mixture introduction
Performing the process such that introducing a phenotypic reporter and disposing it near each nucleic acid sequence occurs after a reagent mixture has been placed near each nucleic acid sequence of the first plurality.
Solution-phase reagent or micro-object/cell/substrate phenotypic reporter
Using a phenotypic reporter that is either a solution-phase reagent providing a detectable signal upon contact with a target protein sequence, or micro-objects with binding sites, reporter cells, and/or enzymatic substrates enabling reporting of protein function or enzymatic activity.
Machine-learning correlation of nucleic acid sequences and phenotypic readouts
Using a first computer component with a machine learning algorithm to correlate individual nucleic acid sequences from a first library with phenotypic readouts from corresponding proximal regions by identifying that the protein function encoded by the nucleic acid sequence is more desirable than the function of the protein sequence of interest.
The claim coverage centers on a microfluidic protein evolution process that couples nucleic-acid libraries with proximal phenotypic reporter readouts and protein expression, followed by identifying nucleic-acid sequences linked to desired phenotypes and determining their nucleotide sequences. Dependent refinements add microfluidic sequestration pen structure, specific DEP electrode activation substrate implementations, options for reporter formats, an ordering constraint between reagent mixture and reporter introduction, and machine-learning-based correlation of sequences with phenotypic readouts.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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