MICROFLUIDIC PLATFORM FOR SYNTHETIC BIOLOGY APPLICATIONS

• US 20120258487A1
• Filed: 04/02/2012
• Published: 10/11/2012
• Est. Priority Date: 04/01/2011
• Status: Active Grant

• Current Assignee: Sandia Corporation (Martin Marietta Corporation), Regents of the University of California (University of California)
• Original Assignee: Sandia Corporation (Martin Marietta Corporation), Regents of the University of California (University of California)
• Inventors: Singh, Anup K., Chandrasekaran, Aarthi, Hillson, Nathan J., Chang, Chieh, Bharadwaj, Rajiv
• Granted Patent: US 10,131,903 B2
• Time in Patent Office: Days
• US Class Current: 435/34
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

This invention provides methods and compositions for assembling biological constructs (e.g., plasmids, transformed cells, etc.). In certain embodiments the methods involve encapsulating separate components of said biological construct each in a fluid droplet confined in a fluid channel; optionally mixing droplets from different fluid channels to for a sequenced order of droplets carrying different components of said biological construct in a channel or chamber; and optionally combining two or more droplets each containing different components of said biological construct to permit said components to react with each other in one or more reactions contributing to the assembly of said biological construct.

Claims

1. A method of assembling a biological construct, said method comprising:

encapsulating separate components of said biological construct each in a fluid droplet confined in a fluid channel;

optionally mixing droplets from different fluid channels to form a sequenced order of droplets carrying different components of said biological construct in a channel or chamber; and

optionally combining two or more droplets each containing different components of said biological construct to permit said components to react with each other in one or more reactions contributing to the assembly of said biological construct.

2. The method of claim 1, wherein said two or more components comprise two or more components independently selected from the group consisting of a promoter, a terminator, a secretion signal, a gene, a vector, and a cell.

3. The method of claim 1, wherein different droplets contain different genes.

4. The method of claim 1, wherein said two or more components react to ligate two or more genes together.

5. The method of claim 1, wherein said two or more components are subject to a restriction digest before or after ligation.

6. The method of claim 1, wherein said two or more components react to ligate a plurality of genes together under control of a promoter.

7. The method of claim 1, wherein two or more components react to introduce said components into a vector.

8. The method of claim 7, wherein said vector is a plasmid or cosmid.

9. The method of claim 1, wherein said two or more components react in a self-assembling nucleic acid assembly system.

10. The method of claim 9, wherein said assembly system comprises a system selected from the group consisting of BioBrick, SLIC, Gibson, CPEC, and Golden-gate.

11. The method of claim 1, wherein said two or more components react to introduce a nucleic acid construct and/or to express a contract in a cell free modality.

12. The method of claim 1, wherein said two or more components react to introduce a nucleic acid construct into a cell.

13. The method of claim 12, wherein said cell is selected from the group consisting of a bacterial cell, a mammalian cell, an insect cell, a plant cell, an algal cell, and a fungal cell.

14. The method of claim 1, wherein said method comprises identifying and optionally capturing/trapping transformed cells.

15. The method of claim 1, wherein said droplets carry reagents sufficient to enable one or more procedures selected from the group consisting of amplification, cloning, expression, and cell transformation.

16. The method of claim 1, wherein said fluid channels are in a microfluidics device.

17. The method of claim 1, wherein said reactions are performed in a microfluidics device.

18. The method of claim 1, wherein the nature and sequence of operations, droplet components, and/or reagent composition of droplets or reaction mixtures is determined and optionally under the control of instructions and/or software implementing instructions according to a j5 assembly protocol.

19. A microfluidic device for the assembly of a biological construct, said microfluidic device comprising:

a configuration of microchannels and/or chambers, and/or ports to generate fluid droplets in a fluid channel and/or a fluid chamber;

a configuration of microchannels and/or chambers or ports to introduce biological materials into said droplets; and

optionally, a configuration of microchannels, and/or chamber, and/or ports, and/or valves to control and determine an order of droplets carrying different biological materials; and

optionally, a configuration of microchannels, and/or chamber, and/or ports, and/or valves to control the combination and reaction of droplets carrying different biological materials and/or to control the reaction of said biological materials with each other.

20. The microfluidic device of claim 19, wherein said device is configured to merge droplets by electrocoalescence.

21. The microfluidic device of claim 19, wherein said device comprises one or more serpentine channels that direct droplets over a plurality of heating pads at different temperatures.

22. The microfluidic device of claim 21 wherein said serpentine channel(s) direct droplets over 4 heating pads each heating pad at a different temperature.

23. The microfluidic device of claim 19, wherein said device comprises microchambers configured to contain cells and to receive a flow of cell culture media.

24. The microfluidic device of claim 19, wherein said device is disposed under a microscope to permit visualization of cells in said device.

25. The microfluidic device of claim 19, wherein said device comprises a plurality of electrodes for detecting cells or droplets and/or for controlling droplet combination and/or for controlling fluid flow.

26. The microfluidic device of claim 19, wherein said device is configured to receive instructions and/or to perform operations that comprise

encapsulating separate components of a biological construct each in a fluid droplet confined in a fluid channel;

optionally mixing droplets from different fluid channels to form a sequenced order of droplets carrying different components of said biological construct in a channel or chamber; and

optionally combining two or more droplets each containing different components of said biological construct to permit said components to react with each other in one or more reactions contributing to the assembly of said biological construct.

27. The microfluidic device of claim 19, wherein said device is configured to receive instructions or software implementing instructions and/or to perform operations according to a j5 assembly protocol.