In vitro evolution in microfluidic systems

• US 20060078888A1
• Filed: 10/08/2004
• Published: 04/13/2006
• Est. Priority Date: 10/08/2004
• Status: Active Grant

• Current Assignee: Harvard University, UK Research and Innovation
• Original Assignee: Harvard University
• Inventors: Link, Darren, Weitz, David, Griffiths, Andrew, Ahn, Keunho, Bibette, Jerome
• Granted Patent: US 7,968,287 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01F 23/41 Emulsifying
• CPC Class Codes: B01F 25/433 Mixing tubes wherein the shape of the tube influences the mixing, e.g. mixing tubes with varying cross-section or provided with inwardly extending profiles
• CPC Class Codes: B01F 25/4331 Mixers with bended, curved, coiled, wounded mixing tubes or comprising elements for bending the flow
• CPC Class Codes: B01F 25/4338 Mixers with a succession of converging-diverging cross-sections, i.e. undulating cross-section
• CPC Class Codes: B01F 33/3011 using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 33/3031 using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00466 in a slurry
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/0052 in the shape of elongated tubes
• CPC Class Codes: B01J 2219/00545 Colours
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00657 One-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/0636 Focussing flows, e.g. to laminate flows
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 : comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 : Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/0415 : electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0487 : fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502746 : characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• 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 3/502776 : specially adapted for focusing or laminating flows
• 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 15/1058 : Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 15/1075 : by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12P 21/00 : Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: G01N 15/1459 : the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/1484 : microstructural devices
• CPC Class Codes: G01N 15/149 : specially adapted for sorting particles, e.g. by their size or optical properties

Abstract

The invention describes a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of: (a) compartmentalising genetic elements into microcapsules; and (b) sorting the genetic elements which express the gene product having the desired activity; wherein at least one step is under microfluidic control. The invention enables the in vitro evolution of nucleic acids and proteins by repeated mutagenesis and iterative applications of the method of the invention.

Claims

1. A method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of:

(a) compartmentalising the genetic elements into microcapsules;

(b) sorting the genetic elements which express gene product (s) having the desired activity;

wherein at least one step is under microfluidic control.

2. A method according to claim 1, wherein at least one step is performed using electronic control of fluidic species.

3. A method according to claim 1, wherein at least one step involves fusion or splitting of microcapsules.

4. A method according to claim 1, comprising the steps of:

(a) compartmentalising the genetic elements into microcapsules;

(b) expressing the genetic elements to produce their respective gene products within the microcapsules; and

(c) sorting the genetic elements which encode gene product (s) having the desired activity.

5. A method according to claim 1, comprising the steps of:

(a) expressing the genetic elements to produce their respective gene products such that the gene products are linked to the genes encoding them;

(b) compartmentalising the genetic elements into microcapsules; and

(c) sorting the genetic elements which encode gene product(s) having the desired activity.

6. A method of claim 1 wherein a gene product with the desired activity induces a change in the microcapsule which enables the microcapsule containing the gene product and the genetic element encoding it to be sorted using microfluid technology.

7. A method according to claim 6 wherein a gene product having the desired activity modifies one or more molecules within the microcapsule which enables the microcapsule containing the gene product and the genetic element encoding it to be sorted.

8. A method according to claim 6 or claim 7 wherein the gene product having the desired activity induces a change in the optical properties of the microcapsule and the microcapsules are sorted thereby.

9. A method according to claim 8, wherein the change in optical properties of the microcapsule is a change in fluorescence.

10. A method according to claim 1, wherein the activity of the desired gene product within the microcapsule results, directly or indirectly, in the modification of the genetic element encoding the gene product to enable the isolation of the genetic element.

11. A method according to claim 10, wherein the expression of a gene product having a desired activity results, directly or indirectly, in the modification of an optical property of a genetic element encoding the gene product and the genetic elements which produce the gene product(s) having the desired activity are sorted according to a change in the optical properties of the genetic elements.

12. A method according to claim 11, wherein the change in the optical properties of the genetic elements is a change in fluorescence.

13. A method according to claim 10, wherein the modification of the genetic element enables it to be further modified outside the microcapsule so as to induce a change in its optical properties.

14. A method according to claim 10 wherein the gene product having the desired activity induces a change in the microcapsule which, when detected, triggers the modification of the genetic element within the compartment to enable the isolation of the genetic element.

15. A method according to claim 14 wherein the change in the microcapsule is a change n the optical properties thereof.

16. A method according to claim 15, wherein the change in optical properties of the microcapsule is a change in fluorescence.

17. A method according to claim 10 wherein a part of the genetic element is a substrate and the activity of the desired gene product within the microcapsule results, directly or indirectly, in the conversion of said substrate into a product which remains part of the genetic element and enables its isolation.

18. A method according to claim 10 wherein a part of the genetic element is a ligand and the desired gene product within the microcapsule binds, directly or indirectly, to said ligand to enable the isolation of the genetic element.

19. A method according to claim 18, wherein the ligand is also encoded by the genetic element.

20. A method according to claim 10, wherein the product of the activity of the desired gene product within the microcapsule results, directly or indirectly, in the generation of a product which is subsequently complexed with the genetic element and enables its isolation.

21. A method according to claim 11, wherein wherein the change in optical properties of the genetic element is due to binding of a gene product with distinctive optical properties to the genetic element.

22. A method according to claim 11, wherein the change in optical properties of the genetic element is due to binding of a ligand with distinctive optical properties by the gene product.

23. A method according to claim 11, wherein the change in optical properties of the genetic element is due to a change in the optical properties of the gene product when bound to ligand.

24. A method according to claim 11, wherein the change in optical properties of the genetic element is due to a change in the optical properties of the ligand when bound by the gene product.

25. A method according to claim 11, wherein the change in optical properties of the genetic element is due to a change in the optical properties of both ligand and gene product on binding.

26. A method according to claim 11, wherein the change in optical properties of the genetic element is due to the different optical properties of the substrate and the product of the reaction being selected.

27. A method according to claim 11, wherein both substrate and product have similar optical properties, but only the product, and not the substrate of the reaction being selected binds to, or reacts with, the genetic element, thereby changing the optical properties of the genetic element.

28. A method according to claim 11, wherein further reagents specifically bind to, or specifically react with, the product (and not the substrate) attached to the genetic element, thereby altering the optical properties of the genetic element.

29. A method according to claim 1 wherein the activity of the desired gene product within the microcapsule results, directly or indirectly, in the alteration of the expression of a second gene within the compartment and the activity of the product of the said second gene enables the sorting of the genetic element.

30. A method according to claim 4, wherein step (b) comprises:

expressing the genetic elements to produce their respective gene products within the microcapsules, linking the gene products to the genetic elements encoding them and isolating the complexes thereby formed.

31. A method according to claim 30, wherein in step (c) the complexes are directly sorted to isolate genetic elements encoding a gene product having the desired activity.

32. A method according to claim 30, wherein in step (c) the complexes are further reacted to induce a conditional change in optical properties of the genetic element dependent on the presence of gene products with the desired activity in the complex.

33. A method according to claim 30, wherein the complexes are subjected to a further compartmentalisation step in order to isolate the genetic elements encoding a gene product having the desired activity.

34. A method according to claim 33 wherein the microcapsules are sorted according to claims 6 to 9.

35. A method according to claim 23 wherein the genetic elements are sorted according to claims 10 to 16.

36. A method according to claim 1, wherein a non-desired activity of a gene product results in a change in the microcapsule or the genetic element which is distinct from that resulting from the desired activity.

37. A method according to claim 36, wherein the change resulting from the non-desired activity is used to negatively select the genetic elements.

38. A method according to claim 36, wherein negative selection is combined with positive selection to improve reaction specificity.

39. A method according to claim 36, wherein the improved reaction specificity is an improvement in binding specificity.

40. A method according to claim 36, wherein the improved reaction specificity is an improvement in regio- and/or stereo-selectivity for substrate and/or product.

41. A method according to claim 36, wherein a non-desired activity of a gene product results in a change in the optical properties of the genetic element which is distinct from that resulting from the desired activity.

42. A method according to claim 41, wherein the optical change resulting from the non-desired activity is used to negatively select the genetic elements.

43. A method according to claim 41, wherein negative selection is combined with positive selection to improve reaction specificity.

44. A method according to claim 41, wherein the improved reaction specificity is an improvement in binding specificity.

45. A method according to claim 41, wherein the improved reaction specificity is an improvement in regio- and/or stereo-selectivity for substrate and/or product.

46. A method according to claim 1, wherein each genetic element encodes more two or more genes and each gene product must have a desired activity in order for the genetic element to to be sorted.

47. A method according to claim 46, wherein each genetic element encodes two or more genes and the gene products must bind to each other in the microcapsule in order for the genetic element to to be sorted.

48. A method according to claim 46, wherein each genetic element encodes more two or more genes and each gene product must have a desired activity in order for the optical properties of the microcapsule or genetic element to be modified and the genetic element to be sorted.

49. A method according to claims 47 and 48, wherein each genetic element encodes two or more genes and the gene products must bind to each other in the microcapsule in order for the optical properties of the microcapsule or genetic element and the genetic element to be sorted.

50. A method according to claim 1, wherein the genetic elements are isolated from a library of genetic elements encoding a repertoire of gene products.

51. A method according to claim 1 wherein, after the genetic elements which encode gene product(s) having the desired activity have been sorted, one or more mutations is introduced into the sorted genetic element (s).

52. A method according to claim 1 further comprising iteratively repeating one or more steps.

53. A method according to claim 1 further comprising amplifying the genetic elements.

54. A method according to claim 1, wherein microencapsulation is achieved by forming a water-in-oil emulsion of the aqueous solution in an oil-based medium.

55. A method according to claim 54, wherein the water-in-oil emulsion is made using a microfluidic system.

56. A method according to claim 1, wherein the emulsion is formed by aqueous droplet break off in a co-flowing steam of an immiscible liquid.

57. A method according to claim 55 or claim 56, wherein the emulsion is formed by subjecting a first fluid surrounded by a second fluid to an electric charge.

58. A method according to claim 1, wherein microcapsules are transported by laminar-flow in a stream of fluid in microfluidic channels.

59. A method according to claim 58 wherein the microcapsules are aqueous microdroplets dispersed in a stream of oil in microfluidic channels.

60. A method according to claim 58 wherein the genetic element in a microcapsule is identified by the relative position of of the microcapsule compared to other microdroplets in a microfluidic channel.

61. A method according to claim 1 wherein the identified microcapsules are sorted using a microfluidic device.

62. A method according to claim 61, wherein the sorting of identified microcapsules is achieved using a microfluidic flow sorting device by steering charged microcapsules using an electric field.

63. A method according to claim 62, wherein the microfluidic device is equipped with a sensor which detects a signal emitted by the microcapsules in the microfluidic channels.

64. A method according to claim 1, wherein microcapsules are fused or split using a microfluidic device.

65. A method according to claim 64, wherein a microcapsule is split by application of an electric field.

66. A method according to claim 64, wherein first and second microcapsules are fused by application of opposite electric charges thereto.

67. A method according to claim 64, wherein microcapsules are fused by the induction of dipoles therein which cause the microcapsules to coalesce.

68. A method according to claim 54, wherein microencapsulation is achieved by forming a water-in-fluorocarbon or water-in-perfluorocarbon emulsion.

69. A method according to claim 54, wherein the fluorocarbon is perfluorooctyl bromide or perfluorooctylethane.

70. A method according to claim 54, wherein the emulsion is formed using F-alkyl dimorpholinophosphate(s).

71. A method according to claim 70, wherein the F-alkyl dimorpholinophosphate(s) have the general formula C_nF₂₊₁C_mH_2mOP(O)[N(CH₂CH₂)₂O]₂.

72. A method according to claim 71 wherein the F-alkyl dimorpholinophosphate is C₈F₁₇C₁₁H₂₂OP(O)[N(CH₂CH₂)₂O]₂.

73. A method according to claim 1, wherein the internal environment of the microcapsules is modified by the addition of one or more reagents to the oil phase.

74. A method according to claim 1, wherein the genetic element comprises the gene attached to a microbead.

75. A method according to claim 1, wherein the microbead is nonmagnetic, magnetic or paramagnetic.

76. A method according to claim 6, wherein the sorting of microcapsules is performed using a microfluidic flow sorting device.

77. A method according to claim 10, wherein the sorting of genetic elements is performed using a microfluidic flow sorting device.

78. A method according to claim 12, wherein the sorting of genetic elements is performed using a fluorescence activated cell sorter (FACS) (or similar device).

79. A method according to claim 8, wherein the change in optical properties of the microcapsule is due to a change in the optical properties of the gene product when bound to ligand.

80. A method according to claim 8, wherein the change in optical properties of the microcapsule is due to a change in the optical properties of the ligand when bound by the gene product.

81. A method according to claim 8, wherein the change in optical properties of the microcapsule is due to a change in the optical properties of both ligand and gene product on binding.

82. A method according to claim 8, wherein the change in optical properties of the microcapsule is due to the different optical properties of the substrate and the product of the reaction being selected.

83. A method according to claims 26 and 82, wherein the different fluorescence properties of the substrate and the product are due to fluorescence resonance energy transfer (FRET).

84. A method according to claim 10, wherein genetic elements modified directly or indirectly by the activity of the desired gene product are further modified by Tyramide Signal Amplification (TSA™

;

NEN), resulting directly or indirectly in a change in the optical properties of said genetic elements thereby enabling their separation.

89. A method according to claim 10, wherein the genetic elements are sorted by affinity purification.

90. A method according to claim 10, wherein the genetic elements are sorted by selective ablation of genetic elements which do not encode the desired gene product.

91. A method according to claim 4, wherein expression of genetic elements to form gene products is initiated by fusing microcapsules containing genetic elements with microcapsules containing reagents for expression.

92. A method according to claim 1 or 85, wherein microcapsules containing genetic elements and the gene products they encode are fused with microcapsules containing a substrate(s) for the reaction to be selected.

93. A method according to claim 1 or 85, wherein microcapsules containing genetic elements and the gene products they encode are fused with microcapsules containing an inhibitor of translation.

94. A method according to claim 92, wherein microcapsules containing genetic elements and the gene products they encode are fused with microcapsules containing a substrate(s) for the reaction to be selected and an inhibitor of translation.

95. A product when isolated according to the method of claim 1 or 85.

85. A method for compartmentalising a genetic element and expressing the genetic element to form its gene product within the compartment, comprising the steps of:

(a) forming an aqueous solution comprising the genetic element and the components necessary to express it to form its gene product;

(b) microencapsulating the solution so as to form a discrete microcapsule comprising the genetic element; and

(c) exposing the microcapsule to conditions suitable for the expression of the genetic element to form its gene product to proceed;

wherein at least one step is under microfluidic control.

86. A method according to claim 85, wherein a library of genetic elements encoding a repertoire of gene products is encapsulated and each genetic element expressed to form its gene product within a microcapsule.

87. A method according to claim 85 or claim 86, wherein microencapsulation is achieved by forming a water-in-oil emulsion of the aqueous solution in an oil-based medium.

88. A microcapsule obtainable by the method of claim 85.

96. A method for preparing a gene product, comprising the steps of:

(a) preparing a genetic element encoding the gene product;

(b) compartmentalising genetic elements into microcapsules;

(c) expressing the genetic elements to produce their respective gene microcapsules;

(c) expressing the genetic elements to produce their respective gene products within the microcapsules;

(d) sorting the genetic elements which produce the gene product (s) having the desired activity; and

(e) expressing the gene product having the desired activity;

wherein at least one step is under microfluidic control.

97. A method for screening a compound or compounds capable of modulating the activity of a gene product, comprising the steps of:

(a) preparing a repertoire of genetic elements encoding gene product;

(b) compartmentalising the genetic elements into microcapsules;

(c) expressing the genetic elements to produce their respective gene products within the microcapsules;

(d) sorting the genetic elements which produce the gene product (s) having the desired activity; and

(e) contacting a gene product having the desired activity with the compound or compounds and monitoring the modulation of an activity of the gene product by the compound or compounds;

wherein at least one step is under microfluidic control.

98. A method for preparing a compound or compounds comprising the steps of:

(a) providing a synthesis protocol wherein at least one step is facilitated by a polypeptide;

(b) preparing genetic elements encoding variants of the polypeptide which facilitates this step;

(c) compartmentalising the genetic elements into microcapsules;

(d) expressing the genetic elements to produce their respective gene products within the microcapsules;

(e) sorting the genetic elements which produce polypeptide gene product (s) having the desired activity; and

(F) preparing the compound or compounds using the polypeptide gene product identified in (e) to facilitate the relevant step of the synthesis;

wherein at least one step is under microfluidic control.

99. A method for preparing a gene product, comprising the steps of:

(a) preparing a genetic element encoding the gene product;

(b) compartmentalising genetic elements into microcapsules;

(c) expressing the genetic elements to produce their respective gene products within the microcapsules;

(d) sorting the genetic elements which produce the gene product (s) having the desired activity using a change in their optical properties; and

(e) expressing the gene product having the desired activity;

wherein at least one step is under microfluidic control.

100. A method for screening a compound or compounds capable of modulating the activity of a gene product, comprising the steps of:

(a) preparing a repertoire of genetic elements encoding gene product;

(b) compartmentalising the genetic elements into microcapsules;

(C) expressing the genetic elements to produce their respective gene products within the microcapsules;

(d) sorting the genetic elements which produce the gene product (s) having the desired activity using a change in their optical properties; and

(e) contacting a gene product having the desired activity with the compound or compounds and monitoring the modulation of an activity of the gene product by the compound or compounds;

wherein at least one step is under microfluidic control.

101. A method for preparing a compound or compounds comprising the steps OF:

(a) providing a synthesis protocol wherein at least one step is facilitated by a polypeptide;

(b) preparing genetic elements encoding variants of the polypeptide which facilitates this step;

(c) compartmentalising the genetic elements into microcapsules;

(d) expressing the genetic elements to produce their respective gene products within the microcapsules;

(e) sorting the genetic elements which produce polypeptide gene product (s) having the desired activity using a change in their optical properties; and

(f) preparing the compound or compounds using the polypeptide gene product identified in (e) to facilitate the relevant step of the synthesis;

wherein at least one step is under microfluidic control.

102. A method for preparing a gene product, comprising the steps of:

(a) preparing a genetic element encoding the gene product;

(b) expressing the genetic elements to produce their respective gene products such that the gene products are linked to the genetic elements encoding them;

(c) compartmentalising genetic elements into microcapsules;

(d) sorting the genetic elements which produce the gene product(s) having the desired activity; and

(e) expressing the gene product having the desired activity;

wherein at least one step is under microfluidic control.

103. A method for screening a compound or compounds capable of modulating the activity of a gene product, comprising the steps of:

(a) preparing a genetic element encoding the gene product;

(b) expressing the genetic elements to produce their respective gene products and linking the gene products to the genetic elements encoding them;

(c) compartmentalising genetic elements into microcapsules;

(d) sorting the genetic elements which produce the gene product(s) having the desired activity; and

(e) contacting a gene product having the desired activity with the compound or compounds and monitoring the modulation of an activity of the gene product by the compound or compounds;

wherein at least one step is under microfluidic control.

104. A method for preparing a compound or compounds comprising the steps of:

(a) providing a synthesis protocol wherein at least one step is facilitated by a polypeptide;

(b) preparing genetic elements encoding variants of the polypeptide which facilitates this step;

(c) expressing the genetic elements to produce their respective gene products such that the gene products are linked to the genetic elements encoding them;

(d) compartmentalising the genetic elements into microcapsules;

(e) sorting the genetic elements which produce polypeptide gene product(s) having the desired activity; and

(f) preparing the compound or compounds using the polypeptide gene product identified in (e) to facilitate the relevant step of the synthesis;

wherein at least one step is under microfluidic control.

105. A method for preparing a gene product, comprising the steps of:

(a) preparing a genetic element encoding the gene product;

(b) expressing the genetic elements to produce their respective gene products;

(c) compartmentalising genetic elements into microcapsules;

(d) sorting the genetic elements which produce the gene product(s) having the desired activity using a change in their optical properties; and

(e) expressing the gene product having the desired activity;

wherein at least one step is under microfluidic control.

106. A method for screening a compound or compounds capable of modulating the activity of a gene product, comprising the steps of:

(a) preparing a genetic element encoding the gene product;

(b) expressing the genetic elements to produce their respective gene products;

(c) compartmentalising genetic elements into microcapsules;

(d) sorting the genetic elements which produce the gene product(s) having the desired activity using a change in their optical properties; and

(e) contacting a gene product having the desired activity with the compound or compounds and monitoring the modulation of an activity of the gene product by the compound or compounds;

wherein at least one step is under microfluidic control.

107. A method for preparing a compound or compounds comprising the steps of:

(a) providing a synthesis protocol wherein at least one step is facilitated by a polypeptide;

(b) preparing genetic elements encoding variants of the polypeptide which facilitates this step;

(c) expressing the genetic elements to produce their respective gene products;

(d) compartmentalising the genetic elements into microcapsules;

(e) sorting the genetic elements which produce polypeptide gene product(s) having the desired activity using a change in their optical properties; and

(f) preparing the compound or compounds using the polypeptide gene product identified in (e) to facilitate the relevant step of the synthesis;

wherein at least one step is under microfluidic control.