# US 2022/0033831 A1 · Systems and methods for high-throughput automated strain generation for non-sporulating fungi > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2022/0033831 A1 - Document ID: US20220033831A1 - Status: Published application - Kind: A1 - Application number: 17/387,634 - Filed: 2021-07-28 - Published: 2022-02-03 - Earliest claimed: 2020-07-31 - Assignees: Zymergen Inc. - Applicants: Zymergen Inc. - Inventors: Benjamin Knox; Nasim Mansoori Zangir; Ryan Evan Martinez; Hassan Abdulla - Web page: https://patentgrep.com/patent/US20220033831A1 ## CPC - C12N 15/80: for fungi - C12N 15/1034: {Isolating an individual clone by screening libraries} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Embodiments disclosed include systems and methods to generate and/or improve non-sporulating filamentous fungal cells using high-throughput techniques. Embodiments disclosed also include systems and methods for evaluation and iterative adjustment of methods to generate and/or isolate and select desired strains of non-sporulating filamentous fungal cells using high-throughput techniques. ## Claims ### Claim 1 A method for isolating clonal transformants from a transformation mixture containing homokaryotic and heterokaryotic transformants, the method comprising: (a) dispensing a discrete volume of a liquid suspension including a predetermined concentration of a DNA source and a predetermined concentration of a protoplast source to a reaction area in a first substrate including a plurality of reaction areas, the dispensing configured to generate, in each reaction area from the plurality of reaction areas, a transformation output in liquid media subject to a one-to-many distribution to a series of growth areas such that a percentage of growth areas, from the series of growth areas, that result in a growth that is derived from the transformation output is below a predetermined threshold value; (b) distributing equal volumes of the transformation output in liquid media from each individual reaction area from the plurality of reaction areas, to the series of growth areas, the series of growth areas being distributed across a plurality of second substrates to spatially separate transformants; and (c) determining, from the series of growth areas, a subset of growth areas that include clonal populations derived from a single transformant. ### Claim 2 (depends on 1) The method of claim 1, wherein the threshold value is approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, or approximately 90%. ### Claim 3 (depends on 1) The method of claim 1, wherein the dispensing results in a probability greater than 0.5 that at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% or all of the growth areas that result in growth include no more than a single transformant. ### Claim 4 (depends on 3) The method of claim 3, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% or all of the growth areas that result in growth include clonal populations that are homokaryotic at a specified locus. ### Claim 5 (depends on 1) The method of claim 1, further comprising: culturing the growth areas, following the distributing the transformation output in each reaction to the series of growth areas, under predefined conditions for a predefined period of time to allow growth of colonies; and screening the series of growth areas for the presence or absence of growth that is derived from a single clonal transformant resulting in a clonal population of a desired strain. ### Claim 6 (depends on 5) The method of claim 5, wherein the screening includes optically distinguishing the presence or absence of growth in the plurality of growth areas. ### Claim 7 (depends on 1) The method of claim 1, wherein the cell is a non-sporulating filamentous fungus. ### Claim 8 (depends on 1) The method of claim 1, wherein the cell is of a fungal strain from the phylum Ascomycota or the phylum Basidiomycota. ### Claim 9 A method for isolating clonal populations derived from single clonal transformants, the method comprising: (a) dispensing a discrete volume of a liquid suspension including a DNA source and a protoplast to each individual reaction area in a first substrate including a plurality of reaction areas, the dispensing configured to generate, in each reaction area from the plurality of reaction areas, a transformation output in liquid media such that the transformation output when subject to a one-to-many distribution to a series of growth areas leads to a percentage of growth areas, from the series of growth areas, that result in a growth that is derived from no more than a single transformant, the percentage being at least one of above or below a predefined threshold value; (b) dispensing, in a high-throughput one-to-many manner, a discrete volume of transformation output in liquid media from each individual reaction area from the plurality of reaction areas, to the series of growth areas distributed across a plurality of second substrates with each second substrate from the plurality of second substrates including a plurality of growth areas mapped to correspond to the plurality of reaction areas included in the first substrate; (c) culturing growth in each growth area of the series of growth areas distributed across the plurality of second substrates; and (d) selecting, from the series of growth areas, a subset of growth areas that include clonal populations derived from a single viable clonal transformant. ### Claim 10 (depends on 9) The method of claim 9, wherein the threshold value is chosen based on an efficacy of transformation associated with the transformation output. ### Claim 11 (depends on 9) The method of claim 9, wherein the dispensing results in a probability greater than 0.5 that no more than at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, or at most 95% or all of the individual growth areas include growth derived from a successful transformant. ### Claim 12 (depends on 9) The method of claim 9, further comprising screening, following the culturing growth in each growth area of the series of growth areas, the series of growth areas for the presence or absence of growth that is derived from a single clonal transformant resulting in a clonal population of a desired strain. ### Claim 13 (depends on 12) The method of claim 12, wherein the screening includes optically distinguishing the presence or absence of growth in the plurality of reactions areas. ### Claim 14 (depends on 9) The method of claim 9, wherein the cell is a non-sporulating filamentous fungus. ### Claim 15 (depends on 9) The method of claim 9, wherein the cell is either an Ascomycete or a Basidiomycete. ### Claim 16 (depends on 9) The method of claim 9, further comprising: diluting, by a predetermined amount, the transformation output in liquid media from each individual reaction area from the plurality of reaction areas, to generate a diluted transformation output from each individual reaction area from the plurality of reaction areas, the dispensing in a high-throughput one-to-many manner at (b) being the dispensing of the diluted transformation output from each individual reaction area from the plurality of reaction areas. ### Claim 17 (depends on 9) The method of claim 9, wherein the dispensing in a high-throughput, one-to-many manner, the discrete volume of transformation output in liquid media from each individual reaction area from the plurality of reaction areas, is done without any dilution of the discrete volume of transformation output in liquid media. ### Claim 18 A high-throughput method for engineering a transformant cell, the method comprising: (a) selecting a set of volumes of liquid suspension including a DNA source sample and a protoplast source associated with the cell, each volume of the liquid suspension from the set of volumes of the liquid suspension being different, by at least one predetermined property, from the remaining volumes of the set of volumes of the liquid suspension; (b) distributing the set of volumes of the liquid suspension across a plurality of reaction areas, each reaction area from the plurality of reaction areas including a discrete quantity of the liquid suspension; (c) transforming the quantity of liquid suspension included in each reaction area from the plurality of reaction areas to generate a transformation output from a set of transformation outputs; (d) distributing the transformation output from the set of transformation outputs in a one-to-many manner to a series of growth areas from a plurality of growth areas; and (e) evaluating the plurality of growth areas to calculate at least one of (i) a number of growth areas that included a successful transformant, or (ii) a number of growth areas that included growth of a clonal population derived from a single clonal transformant. ### Claim 19 (depends on 18) The method of claim 18, wherein the predetermined property is at least one of a volume of DNA source, a concentration of DNA, a molar ratio of specified DNA ends included in the DNA source sample, or a ratio of concentration of DNA source and concentration of protoplast source. ### Claim 20 (depends on 18) The method of claim 18, wherein the predetermined property is a concentration of DNA chosen from concentrations between 1.00× to 5.00×. ### Claim 21 (depends on 18) The method of claim 18, wherein the predetermined property is a dilution chosen from concentrations between 1×/1 to 1×/10. ### Claim 22 (depends on 18) The method of claim 18, further comprising adjusting a composition of the liquid suspension based on the set of transformation outputs to maximize the number of growth areas that included growth of a clonal population derived from a single clonal transformant. ### Claim 23 (depends on 18) The method of claim 18, further comprising screening the set of growth areas to select the volumes of liquid suspension that resulted in a desired range of number of growth areas that included growth of a clonal population derived from a single clonal transformant. ### Claim 24 (depends on 23) The method of claim 23, wherein the screening includes optically distinguishing the presence or absence of growth in the plurality of growth areas. ### Claim 25 (depends on 18) The method of claim 18, wherein the cell is a non-sporulating filamentous fungus. ### Claim 26 (depends on 18) The method of claim 18, wherein the cell is either an Ascomycete or a Basidiomycete. ### Claim 27 A system for high-throughput engineering of a cell, the system comprising: a memory including a set of instructions; a processor coupled to the memory and configured to execute the set of instructions, the set of instructions including instructions to: select a set of volumes of a reaction mixture in liquid media, the reaction mixture including a DNA source and a protoplast source, and each volume of the reaction mixture from the set of volumes of the reaction mixture being selected to be used to generate a transformant output from a set of transformant outputs; receive data associated with the set of transformant outputs generated using the set of dilutions of the reaction mixture in liquid media; receive data associated with a set of growth outputs generated using the set of transformation outputs, each transformation output from the set of transformation outputs being used to generate a series of growth outputs from a plurality of series of growth outputs in a one-to-many manner; and calculate a measure of rate of transformation based on at least one of the data associated with the set of transformant outputs or the data associated with a set of growth outputs. ### Claim 28 (depends on 27) The system of claim 27, the processor being further configured to: determine, based on the measure of rate of transformation, a desired concentration of the reaction mixture to be used for engineering the cell. ### Claim 29 (depends on 27) The system of claim 27, wherein the cell is a non-sporulating filamentous fungus. ### Claim 30 (depends on 27) The system of claim 27, wherein the cell is of a fungal strain from the phylum Ascomycota or the phylum Basidiomycota.