Automated nucleic acid assembly and introduction of nucleic acids into cells

• US 10,557,150 B1
• Filed: 09/14/2019
• Issued: 02/11/2020
• Est. Priority Date: 09/30/2017
• Status: Active Grant

• Current Assignee: Inscripta, Inc.
• Original Assignee: Inscripta, Inc.
• Inventors: Bernate, Jorge, Masquelier, Don, Belgrader, Phillip
• Primary Examiner(s): Hobbs, Michael L
• Application Number:
  US16/571,080
• Time in Patent Office: 150 Days
• CPC Class Codes: C12M 23/44 Multiple separable units; Modules
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12M 41/48 Automatic or computerized control automatic analysis G01N35/00
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

In an illustrative embodiment, automated instruments comprising one or more flow-through electroporation devices or modules are provided to automate transformation of nucleic acids in live cells.

Claims

1. An automated instrument comprising:

a housing configured to house all of some modules;

one or more receptacles configured to receive nucleic acids and cells;

a growth module configured to grow the cells;

a cell concentration module configured to concentrate and render the cells electrocompetent;

a flow-through electroporation (FTEP) module configured to introduce the nucleic acids into the cells;

a selection module configured to select for transformed cells; and

a processor configured to operate the automated instrument based on user input and/or selection of a pre-programmed script; and

an automated liquid handling system to move liquids from the one or more receptacles to the growth module, from the growth module to the cell concentration module, from the cell concentration module to the FTEP module, and from the FTEP module to the selection module, all without user intervention.

2. The automated instrument of claim 1, wherein the FTEP module comprises:

a. a first inlet and a first inlet channel for introducing a fluid comprising cells and nucleic acids into the FTEP module;

b. an outlet and an outlet channel for removing a fluid comprising transformed cells from the FTEP module;

c. a flow channel intersecting and positioned between the first inlet channel and the outlet channel; and

d. two or more electrodes positioned in the flow channel between the intersection of the flow channel with the first inlet channel and the intersection of the flow channel with the outlet channel;

wherein the electrodes apply one or more electric pulses to the cells in the fluid as they pass through the flow channel thereby introducing the nucleic acid into the cells in the fluid.

3. The automated instrument of claim 2 wherein the FTEP module further comprises a reservoir connected to the first inlet for introducing the cells in fluid into the FTEP module and a reservoir connected to the outlet for removing transformed cells from the FTEP module.

4. The automated instrument of claim 2 wherein the FTEP module further comprises a second inlet and a second inlet channel and further comprising a reservoir connected to the second inlet.

5. The automated instrument of claim 4 wherein the second inlet and second inlet channel of the FTEP module are located between the first inlet and first inlet channel and the electrodes of the FTEP module.

6. The automated instrument of claim 4 wherein the second inlet and second inlet channel of the FTEP module are located between the electrodes and the outlet channel and outlet of the FTEP module.

7. The automated instrument of claim 1 wherein the selection module comprises an antibiotic.

8. The automated instrument of claim 1 further comprising a reagent cartridge and the FTEP module is a component of the reagent cartridge.

9. The automated instrument of claim 2 wherein the FTEP module further comprises a filter between the first inlet channel and the electrodes.

10. The automated instrument of claim 1, wherein device is configured for use with bacterial, yeast and mammalian cells.

11. The automated instrument of claim 1, wherein the cell concentration module comprises a filter.

12. An automated instrument comprising:

a housing configured to house all of some modules;

one or more receptacles configured to receive nucleic acids and cells;

a growth module configured to grow the cells;

a cell concentration module configured to concentrate and render the cells electrocompetent;

a flow-through electroporation (FTEP) module configured to introduce the nucleic acids into the cells, wherein the FTEP module comprises a first inlet and a first inlet channel for introducing a fluid comprising the cells and nucleic acids into the FTEP module;

an outlet and an outlet channel for removing a fluid comprising transformed cells from the FTEP module;

a flow channel intersecting and positioned between the first inlet channel and the outlet channel; and

two or more electrodes positioned in the flow channel between the intersection of the flow channel with the first inlet channel and the intersection of the flow channel with the outlet channel, wherein the electrodes apply one or more electric pulses to the cells in the fluid as they pass through the flow channel thereby introducing the nucleic acid into the cells in the fluid, and wherein the FTEP further comprises a filter between the first inlet channel and the electrodes;

a selection module configured to select for transformed cells; and

a processor configured to operate the automated instrument based on user input and/or selection of a pre-programmed script; and

an automated liquid handling system to move liquids from the one or more receptacles to the growth module, from the growth module to the cell concentration module, from the cell concentration module to the FTEP module, and from the FTEP module to the selection module, all without user intervention.

13. The automated instrument of claim 12 wherein the FTEP module further comprises a second inlet and a second inlet channel and further comprising a reservoir connected to the second inlet for introducing the nucleic acids into the FTEP module.

14. The automated instrument of claim 13 wherein the second inlet and second inlet channel of the FTEP module are located between the first inlet and first inlet channel and the electrodes of the FTEP module.

15. The automated instrument of claim 13 wherein the second inlet and second inlet channel of the FTEP module are located between the electrodes and the outlet channel and outlet of the FTEP module.

16. The automated instrument of claim 12 wherein the electrodes of the FTEP module are from 5 mm to 50 cm in diameter.

17. The automated instrument of claim 12 wherein a narrowest part of the flow channel width of the FTEP module is from 10 μ

M to 5 mm.

18. The automated instrument of claim 12, further comprising a reagent cartridge.

19. The automated instrument of claim 18, wherein the FTEP module is a component of the reagent cartridge.

20. The automated instrument of claim 19, wherein the selection module comprises an antibiotic.