US 12,385,038 B2
Systems and methods for identification of optimized protein production and kits therefor
Jason M. McEwen, El Cerrito, CA (US); Troy A. Lionberger, Berkeley, CA (US); Eric K. Sackmann, Emeryville, CA (US); Volker L. S. Kurz, Emeryville, CA (US); and Kellen C. Mobilia, Emeryville, CA (US)
Assigned to Bruker Cellular Analysis, Inc., Emeryville, CA (US)
Filed by BRUKER CELLULAR ANALYSIS, INC., Emeryville, CA (US)
Filed on Apr. 9, 2021, as Appl. No. 17/226,914.
Application 17/226,914 is a continuation of application No. PCT/US2019/055953, filed on Oct. 11, 2019.
Claims priority of provisional application 62/744,578, filed on Oct. 11, 2018.
Prior Publication US 2021/0292751 A1, Sep. 23, 2021
Int. Cl. C12N 15/10 (2006.01); B01L 3/00 (2006.01)
CPC C12N 15/1075 (2013.01) [B01L 3/502761 (2013.01); C12N 15/1065 (2013.01); B01L 2200/0647 (2013.01)] 22 Claims
 
1. A process for evolving a protein, the process comprising:
disposing a first library of nucleic acid sequences within a microfluidic device, wherein the microfluidic device comprises: a housing comprising a base and a microfluidic structure disposed on a surface of the base, wherein the base and the microfluidic structure define a region for holding a first liquid medium and micro-objects suspended therein, and further wherein the microfluidic structure of the microfluidic device comprises a flow path for the first liquid medium and a plurality of chambers; wherein both the flow path and the plurality of chambers are fluidically interconnected with the region; and
further wherein each nucleic acid sequence of the first library comprises one or more variations from a nucleic acid sequence encoding a protein sequence of interest;
introducing a phenotypic reporter into the microfluidic device and disposing it in proximity to each nucleic acid sequence of the first library, wherein 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 into the microfluidic device and disposing it in proximity to each nucleic acid sequence of the first library, wherein the reagent mixture is configured to express a corresponding protein sequence from each nucleic acid sequence of the first library;
expressing the corresponding protein sequence from each nucleic acid sequence of the first library;
detecting the phenotypic readout from a region proximal to one or more nucleic acid sequences of the first library;
identifying individual nucleic acid sequences from the first library having a corresponding proximal region with a desired phenotypic readout; and
determining the nucleotide sequence of the identified nucleic acid sequences of the first library.