Microstructures and use thereof for the directed evolution of biomolecules

• US 20060147909A1
• Filed: 05/31/2002
• Published: 07/06/2006
• Est. Priority Date: 05/31/2001
• Status: Abandoned Application

• Current Assignee: DIREVO Biotech AG (Bayer AG)
• Original Assignee: DIREVO Biotech AG (Bayer AG)
• Inventors: Kettling, Ulrich, Koltermann, Andre, Rarbach, Markus, Stephan, Jens
• Application Number:
  US10/478,809
• Publication Number:
  US 20060147909A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2400/0633 with moving parts
• CPC Class Codes: B01L 2400/0661 shape memory polymer valves
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: C12N 15/1079 Screening libraries by altering the phenotype or phenotypic trait of the host reporter assays C12N15/1086

Abstract

The invention relates to microstructures and the use thereof for the directed evolution of biomolecules.

Claims

1. A method for the cell-free selection of genotype variants from genotype libraries in a microstructure, comprising the following sequence of reaction steps:

(a) combining a test fluid comprising a genotype library in an expressible form and expression aids suitable for cell-free expression with a separation fluid in the microstructure to form individual compartments of the test fluid;

(b) transporting the compartments through the microstructure, the expression of the genotype into the phenotype being effected in the compartments;

(c) detecting the phenotype in the compartments; and

(d) selecting the compartments in accordance with their phenotypes.

21. The method according to claim 1, wherein an assay fluid with reagents suitable for detecting the phenotype is added to the compartments after step (b).

22. The method according to claim 1, wherein said test fluid and expression aids suitable for cell-free expression are selected from aqueous solutions and suspensions of complex compositions.

23. The method according to claim 22, wherein said test fluid contains a cell extract suitable for in-vitro protein expression.

24. The method according to claim 1 wherein said separation fluid is a water-immiscible fluid.

25. The method according to claim 24, wherein the water-immiscible fluid is selected from aliphatic and aromatic hydrocarbons, higher alcohols, higher alkanones, esters and ethers of higher hydrocarbons, halogenated hydrocarbons and silicones and mixtures of these substances.

26. The method according to claim 1 wherein the transport speed of the compartments within the microstructure is from 1×

10<sup>−

7</sup> to 1×

10<sup>−

2</sup> m/s.

27. The method according to claim 26 wherein the transport speed of the compartments with the microstructure is from 1×

10<sup>−

6</sup> to 1×

10<sup>−

4</sup> m/s.

28. The method according to claim 1, wherein the concentration of said genotype library and said combining of the test fluid and the separation fluid are selected in such a way that a statistic average of only one genotype variant is contained per compartment.

29. The method according to claim 1, wherein the compartment volume is from 0.01 fl to 10 pl.

30. The method according to claim 29, wherein the compartment volume is from 0.1 fl to 1 pl.

31. The method according to claim 29, wherein the compartment volume is from 1 to 100 fl.

32. The method according to claim 21, wherein said assay fluid is miscible with said test fluid and immiscible with said separation fluid, and the time of the addition is selected in such a way that an amount of gene product sufficient for detection will already have formed by then.

33. The method according to claim 32 wherein said assay fluid is selected from aqueous solutions, suspensions and emulsions.

34. The method according to claim 1, wherein the assay reagents are specific for the function to be selected.

35. The method according to claim 34, wherein the assay reagents are suitable for analyzing the function to be selected with optical measuring methods.

36. The method according to claim 34, wherein the assay reagent are suitable for analyzing the function to be selected with fluorimetric measuring methods.

37. The method according to claim 1, wherein the detection of the phenotype in the compartments comprises the qualitative determination of the phenotypical properties.

38. The method according to claim 37, wherein the determination of the phenotype is effected by optical methods.

39. The method according to claim 37, wherein the determination of the phenotype properties is effected by fluorimetric methods.

40. The method according to claim 1, wherein the detection of the phenotype in the compartments comprises the quantitative determination of the phenotypical properties.

41. The method according to claim 40, wherein the determination of the phenotype is effected by optical methods.

42. The method according to claim 40, wherein the determination of the phenotype properties is effected by fluorimetric methods.

43. The method according of claim 1, wherein said phenotype is manifested by endonucleolytic activity.

44. The method according of claim 1, wherein said selecting of the compartments is effected by sorting.

45. The method according to claim 44, which further comprises the reaction step of

(e) isolating the genotype of the selected compartments to form a new genotype library.

46. The method according to claim 45, wherein the genotype library obtained is subjected to one or more further reaction cycles (a) to (d).

47. The method according to claim 1 which further comprises the reaction step of

(e) isolating the genotype of the selected compartments to form a new genotype library.

48. The method according to claim 47, wherein the genotype library obtained is subjected to one or more further reaction cycles (a) to (d).

49. A microstructure for performing the method according to claim 1, comprising:

a first supply channel for supplying a test fluid (102) to a reaction channel;

a second supply channel for supplying at least one separation fluid (101) to the reaction channel;

a detection means (205) provided at the end of the reaction channel for detecting a reaction proceeded in the test fluid; and

a selection means for selecting the test fluid compartments (109).

50. The microstructure according to claim 49, wherein the first supply channel is for supplying a fluid containing a genotype.

51. The microstructure according to claim 49, characterized by a first metering means (221, 222) connected with the first or second supply channel for the volume-limited supply of test fluid (102) or separation fluid (101), so that compartments (109, 111) are formed in the reaction channel.

52. The microstructure according to claim 51, characterized by a second metering means (222) which is arranged in such a way that one metering means (221, 222) is provided in each of the first and second supply channels.

53. The microstructure according to claim 52, characterized by a control means connected with said metering means (221, 222) by which said metering means (221, 222) can be controlled in such a way that only test fluid (102) and separation fluid (101) are supplied to the reaction channel alternately.

54. The microstructure according to claim 49, characterized by a third supply channel for supplying assay fluid (103) to the reaction channel.

55. The microstructure according to claim 54, characterized in that a third metering means (223) for the volume-limited supply of assay fluid (103) is connected with the third supply channel and further connected with a recognition means for recognizing a test fluid compartment (109) and can be controlled through a signal transmitted from the recognition means so that the assay fluid (103) is supplied to the test fluid compartment (109).

56. The microstructure according to claim 49, characterized in that said selection means has at least two selection channels (112) connected with the reaction channel, and a selection means (224, 225) for selecting one of the two selection channels (112) depending on the detection result.

57. The microstructure according to claim 56, characterized in that a metering means (224, 225) is provided as a selection means in at least one of said selection channels (112).

58. The microstructure according to claim 49, characterized in that said reaction channels (108, 110) have several individual channels (408, 410) which can be switched in parallel.

59. The microstructure according to claim 58, characterized in that each individual channel has at least one inlet metering means and/or one outlet metering means.

60. The microstructure according to claim 49, characterized in that said metering means (222, 223, 224) are microstructured valve elements.

2-20. -20. (canceled)