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US 2022/0033831 A1Published application
Systems and methods for high-throughput automated strain generation for non-sporulating fungi
AssigneeZymergen Inc.
InventorsBenjamin Knox, Nasim Mansoori Zangir, Ryan Evan Martinez, Hassan Abdulla
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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.
Claims30
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.
1 more element
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.
2 more elements
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.
3 more elements
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.
4 more elements
Dependencies are inferred from the claim text.