Automated nucleic acid assembly and introduction of nucleic acids into cells

Patent metadata

Field	Value
Publication number	US-2019100722-A1
Application number	US-201816147871-A
Country	US
Kind code	A1
Filing date	Sep 30, 2018
Priority date	Sep 30, 2017
Publication date	Apr 4, 2019
Grant date	—

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.

First claim

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 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 cell receptacle to the growth module, from the growth module to the FTEP module, as well as from the one or more nucleic acid receptacles to the FTEP module, all without user intervention.

2 . (canceled)

3 . 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.

4 . 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.

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

8 . The automated instrument of claim 1 wherein the narrowest part of the channel width of the FTEP module is from 10 μM to 5 mm.

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

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

11 . 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 cell receptacle to the growth module, and from the growth module to the FTEP module, as well as from the one or more nucleic acid receptacles to the FTEP module, all without user intervention.

12 . The automated instrument of claim 11 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.

13 . The automated instrument of claim 11 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.

14 . The automated instrument of claim 11 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 11 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 11 wherein the electrodes of the FTEP module are from 5 mm to 50 cm in diameter.

17 . The automated instrument of claim 11 wherein the narrowest part of the channel width of the FTEP module is from 10 μM to 5 mm.

18 . The automated instrument of claim 11 wherein the FTEP module further comprises a filter between the one or more inlet channels and the electrodes.

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

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

Assignees

• Inscripta Inc

Inventors

• Bernate Jorge
• Masquelier Don
• Belgrader Phillip

Classifications

• C12N15/87 Primary

  Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation · CPC title

• C12N15/81

  for yeasts · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12M35/02 Primary

  Electrical or electromagnetic means, e.g. for electroporation or for cell fusion · CPC title

• C12M41/48

  Automatic or computerized control (automatic analysis G01N35/00) · CPC title