Automated nucleic acid assembly and introduction of nucleic acids into cells

• US 10,415,058 B2
• Filed: 09/30/2018
• Issued: 09/17/2019
• 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): Bowers, Nathan A
• Application Number:
  US16/147,871
• Publication Number:
  US 20190100722A1
• Time in Patent Office: 352 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 of the modules;

a receptacle configured to receive cells;

one or more receptacles configured to receive nucleic acids;

a growth module in which to grow the cells;

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

wherein the FTEP module comprises;

a. one or more inlets and one or more inlet channels for introducing a fluid comprising cells and nucleic acid 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 a first inlet channel and the outlet channel, wherein the flow channel decreases in width between the first inlet channel and the center of the flow channel and the outlet channel and the center of the flow 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 are in fluid communication with fluid in the flow channel but are not in the direct flow path of the cells in the flow channel; and

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;

a recovery module configured to allow the cells to recover after cell transformation in the FTEP module;

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 cells receptacle configured to receive cells to the growth module, from the growth module to the FTEP module, as well as from the one or more receptacles configured to receive nucleic acids to the FTEP module, all without user intervention.

2. The automated instrument of claim 1 wherein the FTEP module further comprises a reservoir connected to the 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.

3. The automated instrument of claim 1 wherein the FTEP module further comprises two inlets and two inlet channels and further comprising a reservoir connected to a second inlet for introducing the nucleic acid into the FTEP module.

4. The automated instrument of claim 3 wherein the second inlet and a second inlet channel of the FTEP module are located between the first inlet and first inlet channel and the electrodes of the FTEP module.

5. The automated instrument of claim 3 wherein the second inlet and a second inlet channel of the FTEP module are located between the electrodes and the outlet channel and outlet of the FTEP module.

6. The automated instrument of claim 1 wherein the electrodes of the FTEP module are from 5 mm to 50 cm in diameter.

7. The automated instrument of claim 1 wherein the narrowest part of the channel width of the FTEP module is from 10 μ

M to 5 mm.

8. The automated instrument of claim 1 wherein the FTEP module further comprises a filter between the one or more inlet channels and the electrodes.

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

18. The automated instrument of claim 1, further comprising a reagent cartridge.

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

10. An automated instrument comprising an FTEP module, wherein the automated instrument comprises:

a housing configured to house all of some of the modules;

a receptacle configured to receive cells;

a growth module for growing the cells;

one or more receptacles configured to receive nucleic acids;

the flow-through electroporation (FTEP) module, wherein the FTEP module is configured to introduce the nucleic acids into the cells; and

wherein the FTEP module comprises;

a. at least one inlet and at least one inlet channel for introducing a fluid comprising cells and nucleic acid to the FTEP module;

b. an outlet and an outlet channel for removing transformed cells and nucleic acid from the FTEP module;

c. a flow channel positioned between a first inlet channel and the outlet channel, wherein the flow channel intersects with the first inlet channel and the outlet channel and wherein a portion of the flow channel narrows between the inlet channel intersection and the outlet channel intersection; and

d. an electrode positioned on either side of the flow channel and in direct contact with the fluid in the flow channel, the electrodes defining the narrowed portion of the flow channel; and

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;

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 receptacle configured to receive cells to the growth module, and from the growth module to the FTEP module, as well as from the one or more receptacles configured to receive nucleic acids to the FTEP module, all without user intervention.

11. The automated instrument of claim 10 wherein the FTEP module further comprises a reservoir connected to the 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.

12. The automated instrument of claim 10 wherein the FTEP module further comprises two inlets and two inlet channels and further comprising a reservoir connected to a second inlet for introducing the nucleic acid into the FTEP module.

13. The automated instrument of claim 12 wherein the second inlet and a second inlet channel of the FTEP module are located between the first inlet and first inlet channel and the electrodes of the FTEP module.

14. The automated instrument of claim 12 wherein the second inlet and a second inlet channel of the FTEP module are located between the electrodes and the outlet channel and outlet of the FTEP module.

15. The automated instrument of claim 10 wherein the electrodes of the FTEP module are from 5 mm to 50 cm in diameter.

16. The automated instrument of claim 10 wherein the narrowest part of the channel width of the FTEP module is from 10 μ

M to 5 mm.

17. The automated instrument of claim 10 wherein the FTEP module further comprises a filter between the one or more inlet channels and the electrodes.