Automated control of cell growth rates for induction and transformation

a cell growth rate and induction technology, applied in biochemical apparatus and processes, specific use bioreactors/fermenters, after-treatment of biomass, etc., can solve the problems of contaminated cell culture, time and effort required for procedures, and human intervention

Active Publication Date: 2019-10-03
INSCRIPTA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020]These aspects and other features and advantages of the invention are described below in more detail.

Problems solved by technology

One drawback to using this traditional method for measuring OD is that it requires human intervention; that is, aliquots of the sample to be measured must be taken at intervals, loaded into cuvettes, and inserted into the spectrophotometer to get a reading.
Not only does this procedure require time and effort, but invasively accessing the growing cell culture runs a risk that the cell culture may be contaminated.
Further, the cell culture is depleted with each sample removed.
An additional drawback is that once the cells are growing, it is difficult to predict when the cells will reach a target OD.

Method used

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  • Automated control of cell growth rates for induction and transformation
  • Automated control of cell growth rates for induction and transformation
  • Automated control of cell growth rates for induction and transformation

Examples

Experimental program
Comparison scheme
Effect test

example i

Growth in the Cell Growth Module

[0113]One embodiment of the cell growth device as described herein was tested against a conventional cell shaker shaking a 5 ml tube and an orbital shaker shaking a 125 ml baffled flask to evaluate cell growth in bacterial and yeast cells. Additionally, growth of a bacterial cell culture and a yeast cell culture was monitored in real time using an embodiment of the cell growth device described herein.

[0114]In a first example, 20 ml EC23 cells (E. coli cells) in LB were grown in a 35 ml rotating growth vial with a 2-paddle configuration at 30° C. using the cell growth device as described herein. The rotating growth vial was spun at 600 rpm and oscillated (i.e., the rotation direction was changed) every 1 second. In parallel, 5 ml EC23 cells in LB were grown in a 5 ml tube at 30° C. and were shaken at 750 rpm. OD600 was measured at intervals using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific). The results are shown in FIG. 8. The rotating gro...

example ii

Fully-Automated Singleplex RGN-directed Editing Run

[0118]Singleplex automated genomic editing using MAD7 nuclease was successfully performed with an automated multi-module instrument of the disclosure. See U.S. Pat. No. 9,982,279.

[0119]An ampR plasmid backbone and a lacZ_F172* editing cassette were assembled via Gibson Assembly® into an “editing vector” in an isothermal nucleic acid assembly module included in the automated instrument. lacZ_F172 functionally knocks out the lacZ gene. “lacZ_F172*” indicates that the edit happens at the 172nd residue in the lacZ amino acid sequence. Following assembly, the product was de-salted in the isothermal nucleic acid assembly module using AMPure beads, washed with 80% ethanol, and eluted in buffer. The assembled editing vector and recombineering-ready, electrocompetent E. Coli cells were transferred into a transformation module for electroporation. The cells and nucleic acids were combined and allowed to mix for 1 minute, and electroporation w...

example iii

Fully-Automated Recursive Editing Run

[0122]Recursive editing was successfully achieved using the automated multi-module cell processing system. An ampR plasmid backbone and a lacZ_V10* editing cassette were assembled via Gibson Assembly® into an “editing vector” in an isothermal nucleic acid assembly module included in the automated system. Similar to the lacZ_F172 edit, the lacZ_V10 edit functionally knocks out the lacZ gene. “lacZ_V10” indicates that the edit happens at amino acid position 10 in the lacZ amino acid sequence. Following assembly, the product was de-salted in the isothermal nucleic acid assembly module using AMPure beads, washed with 80% ethanol, and eluted in buffer. The first assembled editing vector and the recombineering-ready electrocompetent E. Coli cells were transferred into a transformation module for electroporation. The cells and nucleic acids were combined and allowed to mix for 1 minute, and electroporation was performed for 30 seconds. The parameters fo...

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Abstract

The present disclosure relates to methods and devices for automated control of cell growth rates where cell growth is measured in situ and the devices can be used as a stand-alone device or as a module in an automated environment, e.g., as one module in a multi-station or multi-module cell processing environment. The cell growth device comprises a temperature-controlled vial, a motor assembly to spin the vial, a spectrophotometer for measuring, e.g., OD of the cells in the vial, and a processor to accept input from a user and control the growth rate of the cells.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present application claims priority to U.S. Provisional Patent Application No. 62 / 649,731, filed Mar. 29, 2018, and U.S. Provisional Patent Application No. 62 / 671,385, filed May 14, 2018 and are both incorporated herein by reference.FIELD OF THE INVENTION[0002]The present disclosure relates to methods, devices, and instruments for automated control of cell growth rates and subsequent cell processing. The devices can be used as stand-alone cell growth devices or as one module in an automated cell processing environment.BACKGROUND OF THE INVENTION[0003]In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.[0004]...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12M1/42C12M1/00C12N15/87
CPCC12M35/02C12M29/10C12M35/08C12M35/04C12N15/87C12M41/36C12M41/48C12M27/14C12M23/08C12M23/44C12M27/02
Inventor MASQUELIER, DONBELGRADER, PHILLIP
Owner INSCRIPTA INC
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