Microfluidic platform for synthetic biology applications

Patent metadata

Field	Value
Publication number	US-10131903-B2
Application number	US-201213437727-A
Country	US
Kind code	B2
Filing date	Apr 2, 2012
Priority date	Apr 1, 2011
Publication date	Nov 20, 2018
Grant date	Nov 20, 2018

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

First claim

What is claimed is:

1. A method of assembling a biological construct, said method comprising:
encapsulating a plurality of separate components of said biological construct in discrete and separate fluid droplets to provide different components of said construct each in different discrete fluid droplets, where said discrete fluid droplets are disposed in an immiscible fluid that separates said droplets, where a plurality of discrete fluid droplets containing the same type of component are in a single fluid channel containing said immiscible fluid and a plurality of single fluid channels are disposed in a microfluidics device to provide a microfluidics device comprising different fluid channels where each fluid channel contains a plurality of discrete fluid droplets each containing the same type of component and the fluid droplets in different fluid channels contain different components;
introducing fluid droplets from said different fluid channels of said device into a common channel of said device in fluid communication with said plurality of fluid channels to form a spatially sequenced order of separate discrete fluid droplets in an immiscible fluid inside said common channel where different droplets comprising said spatially sequenced order of discrete fluid droplets contain said different components; and
moving the immiscible fluid containing said separate discrete fluid droplets along said common channel and into a droplet merging channel in fluid communication with said common channel to combine two or more fluid droplets of said spatially sequenced order of fluid droplets into a common droplet in said immiscible fluid and to react said separate components with each other in one or more reactions that assemble said separate components together to form said biological construct.

2. The method of claim 1 , wherein said separate components of said biological construct comprise 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 2 , wherein said separate components each contain an element independently selected from the group consisting of a promoter, a terminator, a secretion signal, a gene, and a vector, and said combining two or more fluid droplets of said sequenced order of fluid droplets to react said separate components with each other comprises combining two or more fluid droplets wherein at least one of said fluid droplets further contains a restriction enzyme whereby said combining performs a restriction digest reaction in the combined fluid droplets.

4. The method of claim 1 , wherein said separate components of said biological construct comprise genes and different fluid droplets comprising said sequenced order of fluid droplets contain different genes.

5. The method of claim 4 , wherein said combining two or more fluid droplets of said sequenced order of fluid droplets to react said separate components with each other comprises combining two or more fluid droplets wherein at least one of said two or more fluid droplets further contains a ligase whereby said combining performs a ligation of said different genes to each other in the combined fluid droplets.

6. The method of claim 1 , wherein said combining two or more fluid droplets of said sequenced order of fluid droplets to react said separate components with each other comprises combining two or more fluid droplets wherein at least one of said two or more fluid droplets further contains a ligase whereby said combining performs a ligation of a plurality of genes together under the control of a promoter in the combined fluid droplets.

7. The method of claim 1 , wherein said combining two or more fluid droplets of said sequenced order of fluid droplets to react said separate components with each other comprises combining two or more fluid droplets, wherein at least one of said two or more fluid droplets further contains a vector and said combining introduces said components into said vector.

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

9. The method of claim 1 , wherein said combining two or more fluid droplets of said sequenced order of fluid droplets to react said separate components with each other comprises combining two or more fluid droplets to assemble a nucleic acid construct using constructs selected from the group consisting of DNA sequences that conform to a restriction-enzyme assembly standard, constructs for sequence and ligation independent cloning (SLIC), constructs configured for the joining of multiple DNA fragments in a single, isothermal reaction, constructs for circular polymerase extension cloning (CPEC), and constructs incorporating a recognition site for a single type IIS restriction enzyme and a T4 DNA ligase.

10. The method of claim 1 , wherein said combining two or more fluid droplets of said sequenced order of fluid droplets to react said separate components with each other comprises combining two or more fluid droplets, wherein at least one of said two or more fluid droplets further contains a cell and said combining introduces a nucleic acid construct into said cell.

11. The method of claim 10 , 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.

12. The method of claim 10 , wherein said method further comprises identifying and capturing and/or trapping a cell containing said construct.

13. The method of claim 1 , wherein said immiscible fluid comprises an oil.

Assignees

• Chang Chieh
• Bharadwaj Rajiv
• Singh Anup K
• Chandrasekaran Aarthi
• Hillson Nathan J
• Univ California
• Sandia Corp

Inventors

• Chang Chieh
• Bharadwaj Rajiv
• Singh Anup K
• Chandrasekaran Aarthi
• Hillson Nathan J

Classifications

• C12N15/10

  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 title

• C12N15/66

  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 · CPC title

• C12N15/1027 Primary

  by DNA shuffling, e.g. RSR, STEP, RPR · CPC title