SYSTEMS AND METHODS FOR AMPLIFICATION AND PHAGE DISPLAY

• US 20130210680A1
• Filed: 06/10/2011
• Published: 08/15/2013
• Est. Priority Date: 06/10/2010
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Derda, Ratmir, Whitesides, George M., Tang, Sindy K.Y.
• Granted Patent: US 9,499,813 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

The present invention generally relates to amplification of biological entities, for example, for phage display. In one aspect, members of a library of biological entities are encapsulated in separate compartments (e.g., in separate microfluidic droplets) and amplified. As a specific example, by putting members of a phage display library into microfluidic droplets such that no droplet contains more than one member of the library, the library can be amplified without any substantial changes in growth rates or population distributions, or other artifacts created due to differences in growth rates or amplification between different members of the library. In some cases, the volume of the compartments can be used to control the copy number of a biological entity during amplification. In certain cases, biological entities with different amplification rates can be amplified independently of each other. In some embodiments, the ratio of a rapidly amplifying biological entity to a slowly amplifying biological entity can be controlled. This can be advantageous, for example, in preserving diversity within a library by preventing rapidly amplifying biological entities from outcompeting slowly amplifying biological entities. For example, certain methods and systems of the invention can be useful in situations where preferential amplification of library members can present a problem.

Claims

1. A method of producing an amplified library of clones, the method comprising:

a. distributing a library of clones comprising a plurality of distinguishable replicable genetic package members into a plurality of monodisperse individual compartments such that substantially no more than one replicable genetic package member is contained in any individual compartment; and

b. amplifying the library of step (a) for a sufficient period of time such that each of the distinguishable members replicates to reach substantially the same copy number within the individual compartments;

thereby maintaining diversity of the library of clones upon amplification.

2. The method of claim 1, wherein the replicable genetic package is a virus, a eukaryotic cell, a spore, a yeast cell, a bacterial cell, a nucleic acid sequence, or a recombinant thereof.

3. The method of claim 1, wherein the replicable genetic package is a virus, and wherein substantially each of the individual compartments further comprises at least one host cell capable of supporting replication of the replicable genetic package, wherein the number of host cells within each of the individual compartments is substantially the same.

4. The method of claim 3, wherein substantially each of the individual compartments further comprises at least ten host cells capable of supporting replication of the replicable genetic package.

5. The method of claim 3, wherein the virus is a bacteriophage and the host cells are bacterial cells.

6. The method of claim 1, wherein each of the individual compartments further comprises growth or replication media containing substantially the same amounts of nutrients.

7. The method of claim 1, wherein each of the distinguishable members replicates to reach saturation.

8. The method of claim 7, wherein the saturation is reaching a maximum copy number allowed in the individual compartments.

9. The method of claim 1, wherein the individual compartments are fluidic droplets.

10. The method of claim 1, wherein the plurality of distinguishable replicable genetic package members are distributed into a plurality of fluidic droplets using at least one microfluidic device.

11. The method of claim 9, wherein the fluidic droplets are suspended in a perfluorinated liquid and stabilized by at least one surfactant.

12. The method of claim 1, further comprising the step of releasing the amplified distinguishable members within the individual compartments into one common fluid.

13. The method of claim 12, wherein the step of releasing is performed by adding at least one destabilization agent into the amplified library of step (b).

14. The method of claim 12, wherein the step of releasing is performed by diluting the at least one surfactant.

15. The method of claim 12, wherein the step of releasing is performed in the presence of electric fields.

16. The method of claim 1, wherein the monodisperse compartments have an average diameter of at least about 40 μ

m.

17. The method of claim 1, wherein the monodisperse compartments have an average diameter of about 20 μ

m to about 500 μ

m.

18. The method of claim 1, wherein the monodisperse compartments have an average diameter of no larger than 200 μ

m.

19. The method of claim 1, wherein the library comprises at least about 100 distinguishable clones.

20. The method of claim 1, wherein the library comprises at least about 1,000 distinguishable clones.

21. The method of claim 1, wherein the library comprises at least about 10⁶ distinguishable clones.

22. The method of claim 1, wherein the library comprises at least about 10⁹ distinguishable clones.

23. The method of claim 1, wherein the amplified library of clones is selected from the group consisting of phage display, yeast display, bacterial display, RNA display, DNA display, and ribosome display.

24. A method of producing an amplified library of phage clones, the method comprising:

a. distributing a library of phage clones comprising a plurality of distinguishable phage clones into a plurality of monodisperse individual compartments such that substantially no more than one phage clone is contained in any individual compartment, and wherein each of the individual compartments further comprises at least one bacterial cell; and

b. culturing the library of step (a) for a sufficient period of time such that the bacterial cells replicate essentially to the same number within the individual compartments, thereby producing substantially the same copy number of each phage clone within the individual compartments;

thereby maintaining diversity of the library of phage clones upon amplification.

25-61. -61. (canceled)

62. A method of producing an amplified library of viral clones, the method comprising:

providing an initial library of replicable genetic packages having a first distribution of growth rates including a first mean and a first standard deviation; and

amplifying the initial library of replicable genetic packages to produce an amplified library of replicable genetic packages having a second distribution of growth rates including a second mean and a second standard deviation, wherein the first mean and the second mean differ by no more than about 10% relative to the first mean and the first standard deviation and the second standard deviation differ by no more than about 10% relative to the first standard deviation.

63-90. -90. (canceled)