received by the International Bureau on 6 February 2018 (06.02.2018)

1. An engineered bacterium comprising:

a. an orthogonal translation system comprising a heterologous gene encoding an orthogonal aminoacyl tRNA synthetase and a heterologous gene encoding an orthogonal tRNA; and

b. a heterologous nucleic acid comprising a labeling codon, wherein the heterologous nucleic acid comprises a heterologous gene encoding a protein selected from the group consisting of a secreted protein, a membrane protein, or a bacterial extracellular matrix protein;

wherein the aminoacyl tRNA synthetase is capable of specifically aminoacylating the orthogonal tRNA with a nonstandard amino acid; and

wherein the orthogonal tRNA incorporates the nonstandard amino acid at the residue corresponding to the labeling codon during translation.

2. The engineered bacterium of claim 1, wherein the heterologous nucleic acid further encodes a polypeptide linker sequence.

3. The engineered bacterium of claim 2, wherein the polypeptide linker sequence is flexible.

4. The engineered bacterium of any one of claims 1-3, wherein the heterologous nucleic acid further encodes a polypeptide tag.

5. The engineered bacterium of any one of claims 1-4, wherein the labeling codon is located at a nucleic acid position within the heterologous gene encoding the protein.

6. The engineered bacterium of any one of claims 2-4, wherein the labeling codon is located at a nucleic acid position within the heterologous nucleic acid encoding the polypeptide linker sequence.

7. The engineered bacterium of any one of claims 2-4, wherein the labeling codon is located at a nucleic acid position within the heterologous nucleic acid encoding the polypeptide tag.

8. The engineered bacterium of any one of claims 1-7, wherein the heterologous nucleic acid comprises 2, 3, 4, 5, 6, 7, or 8 labeling codons.

9. An engineered bacterium comprising:

a. an orthogonal translation system comprising a heterologous gene encoding an orthogonal aminoacyl tRNA synthetase and a heterologous gene encoding an orthogonal tRNA; and

b. a heterologous nucleic acid encoding a protein, wherein the protein comprises a nonstandard amino acid, and wherein the protein is selected from the group consisting of a secreted protein, a membrane protein, or a bacterial extracellular matrix protein;

wherein the aminoacyl tRNA synthetase is capable of specifically aminoacylating the orthogonal tRNA with the nonstandard amino acid; and

wherein the orthogonal tRNA incorporates the nonstandard amino acid into the protein during translation.

10. The engineered bacterium of claim 9, wherein the protein further comprises a polypeptide linker.

1 1. The engineered bacterium of claim 10, wherein the polypeptide linker is flexible.

12. The engineered bacterium of any one of claims 9-1 1, wherein the protein further comprises a polypeptide tag.

13. The engineered bacterium of claim 4 or claim 12, wherein the polypeptide tag is selected from the group consisting of a poly-histidine tag, a myc tag, a FLAG tag, a hemagglutinin (HA) tag, and a V5 tag.

14. The engineered bacterium of any one of claims 9-13, wherein the protein comprises 2, 3, 4, 5, 6, 7, or 8 nonstandard amino acids.

15. The engineered bacterium of any one of claims 1- 14, wherein the bacterial extracellular matrix protein is a fiber protein, a flagellar protein, or a pilin protein.

16. The engineered bacterium of any one of claims 1-14, wherein the protein is selected from the group consisting of InaV, Trat, and CsgA.

17. The engineered bacterium of claim 16, wherein the protein is CsgA.

18. The engineered bacterium of any one of claims 9-17, wherein the protein is CsgA and the nonstandard amino acid is located at an amino acid residue corresponding to residue 89 of wild-type CsgA.

19. The engineered bacterium of any one of claims 9-16, wherein the protein is InaV and the nonstandard amino acid is located at an amino acid residue corresponding to residue 174 of wild-type InaV.

20. The engineered bacterium of any one of claims 9-16, wherein the protein is Trat and the nonstandard amino acid is located at an amino acid residue corresponding to residue 200 of wild-type Trat.

21. The engineered bacterium of any one of claims 1-8, wherein the labeling codon is TAG.

22. The engineered bacterium of claim 1 or claim 9, wherein the orthogonal tRNA comprises an anticodon loop that specifically recognizes the codon UAG.

23. The engineered bacterium of any one of claims 1-22, wherein the nonstandard amino acid comprises a bioorthogonal reactive functional group.

24. The engineered bacterium of claim 23, wherein the bioorthogonal reactive functional group is selected from the group consisting of an azide moiety, a ketone moiety, an alkyne moiety, an alkene moiety, a tetrazine moiety, or a norbornene moiety.

25. The engineered bacterium of any one of claims 1-24, wherein the nonstandard amino acid is selected from the group consisting ofp-azidophenylalanine (p-AzF),

azidohomoalanine (Aha), azidolysine, homopropargylglycine (Hpg), homoallylglycine (Hag), oxonorvaline (Onv), /?-bromophenylalanine (p-BrF), /?-iodophenylalanine {p-l¥), p- ethynylphenylalanine (p-EtF), /7ora-acetylphenylalanine (p-AcF), azidonorleucine (Anl), irara-crotylglycine (Teg), selenomethionine (Se-Met), 2-aminooctynoic acid (Aoa), and propargylglycine (Pra).

26. The engineered bacterium of claim 25, wherein the nonstandard amino acid is p-AzV.

27. The engineered bacterium of any one of claims 1-26, wherein the engineered bacterium is non-pathogenic.

28. The engineered bacterium of any one of claims 1-27, wherein the engineered bacterium is of the species Escherichia coli.

29. The engineered bacterium of any one of claims 1-28, wherein the engineered bacterium is of an Escherichia coli strain selected from the group consisting of C321 , C321.AA and C321.AA.exp.

30. The engineered bacterium of any one of claims 1-28, wherein the engineered bacterium is of the Escherichia coli Nissle 1917 strain.

31. The engineered bacterium of any one of claims 1-30, wherein the engineered bacterium comprises a genome that has been recoded to reduce or eliminate a codon.

32. The engineered bacterium of claim 31 , wherein the engineered bacterium comprises a genome that has been recoded to reduce or eliminate a TAG codon.

33. The engineered bacterium of any one of claims 1-30, wherein the engineered bacterium comprises a genome that has not been recoded to reduce or eliminate a codon.

34. The engineered bacterium of any one of claims 1-33, wherein the engineered bacterium further comprises a prfA gene deletion.

35. The engineered bacterium of any one of claims 1-34, wherein the orthogonal aminoacyl tRNA synthetase is an Archea aminoacyl tRNA synthetase or a bacterial aminoacyl tRNA synthetase.

36. The engineered bacterium of any one of claims 1-35, wherein the heterologous gene encoding the orthogonal aminoacyl tRNA synthetase is a Methanosarcina,

Desufitobacterium, Pyrococcus or Methanocaldococcus aminoacyl tRNA synthetase gene.

37. The engineered bacterium of any one of claims 1-36, wherein the heterologous gene encoding the orthogonal aminoacyl tRNA synthetase is a Methanosarcina mazei,

Methanosarcina barkeri, Desulfitobacterium hafniense, Pyrococcus horikoshii, or Methanocaldococcus jannaschii aminoacyl tRNA synthetase gene.

38. The engineered bacterium of any one of claims 1-37, wherein the orthogonal translation system is located in the bacterial genome.

39. The engineered bacterium of any one of claims 1-37, wherein the orthogonal translation system is located in a plasmid.

40. The engineered bacterium of claim 39, wherein the plasmid is pEVOL-pAzF.

41. The engineered bacterium of any one of claims 1-40, wherein the expression of the heterologous nucleic acid is under the control of a constitutive promoter.

42. The engineered bacterium of any of claims 1 -40, wherein the expression of the heterologous nucleic acid is under the control of an inducible promoter.

43. The engineered bacterium of claim 42, wherein the inducible promoter is responsive to an inducer selected from the group consisting of IPTG, arabinose, and tetracycline.

44. The engineered bacterium of any one of claims 1-43, wherein the expression of the heterologous gene encoding the orthogonal aminoacyl tRNA synthetase and/or the heterologous gene encoding the orthogonal tRNA is under the control of a constitutive promoter.

45. The engineered bacterium of any one of claims 1-43, wherein the expression of the heterologous gene encoding the orthogonal aminoacyl tRNA synthetase and/or the heterologous gene encoding the orthogonal tRNA is under the control of an inducible promoter.

46. The engineered bacterium of claim 45, wherein the inducible promoter is responsive to an inducer selected from the group consisting of IPTG, arabinose, and tetracycline.

47. A biofilm comprising the engineered bacterium of any one of claims 1-46.

48. A pharmaceutical composition comprising an engineered bacterium of any one of claims 1-46 and a pharmaceutically-acceptable excipient.

49. The pharmaceutical composition of claim 48, wherein the composition is formulated for oral administration to a subject.

50. The pharmaceutical composition of claim 48, wherein the composition is formulated for rectal administration to a subject.

51. The pharmaceutical composition of claim 49 or claim 50, wherein the subject is a mammalian subject.

52. The pharmaceutical composition of claim 51, wherein the mammalian subject is a human subject.

53. The pharmaceutical composition of any one of claims 48-52, wherein the pharmaceutical composition is formulated as a pill, a capsule, a lozenge, or a suppository.

54. A method for labeling of an engineered bacterium in a subject in vivo, the method comprising:

(a) administering to the subject an engineered bacterium comprising a protein having at least one nonstandard amino acid, wherein said nonstandard amino acid comprises a first bioorthogonal reactive functional group; and

(b) administering to the subject a detectable compound comprising a second bioorthogonal reactive functional group,

wherein the first bioorthogonal reactive functional group reacts with the second bioorthogonal reactive functional group in vivo, thereby attaching the detectable compound to the nonstandard amino acid of the protein,

thereby labeling the engineered bacterium in the subject in vivo.

55. A method for detecting the distribution of an engineered bacterium in a subject in vivo, the method comprising:

(a) administering to the subject an engineered bacterium comprising a protein having at least one nonstandard amino acid, wherein said nonstandard amino acid comprises a first bioorthogonal reactive functional group;

(b) administering to the subject a detectable compound comprising a second bioorthogonal reactive functional group, wherein the first bioorthogonal reactive functional group reacts with the second bioorthogonal reactive functional group in vivo, thereby attaching the detectable compound to the nonstandard amino acid; and

(c) detecting the detectable compound in the subject,

thereby detecting the distribution of the engineered bacterium in the subject in vivo.

56. A method for diagnosing a gastrointestinal disease in a subject, the method comprising

(a) administering to the subject an engineered bacterium comprising a protein having at least one nonstandard amino acid, wherein said nonstandard amino acid comprises a first bioorthogonal reactive functional group;

(b) administering to the subject a detectable compound comprising a second bioorthogonal reactive functional group, wherein the first bioorthogonal reactive functional group reacts with the second bioorthogonal reactive functional group in vivo, thereby attaching the detectable compound to the nonstandard amino acid; and (c) detecting the distribution of the detectable compound in the gastrointestinal tract of the subject, thereby diagnosing a gastrointestinal disease in the subject.

57. A method for detecting the distribution of a biofilm produced by an engineered bacterium in a subject, the method comprising:

(a) administering to the subject an engineered bacterium comprising a protein having at least one nonstandard amino acid to a subject, and wherein the nonstandard amino acid comprises a first bioorthogonal reactive functional group;

(b) administering to the subject a detectable compound comprising a second bioorthogonal reactive functional group, wherein the first bioorthogonal reactive functional group reacts with the second bioorthogonal reactive functional group in vivo, thereby attaching the detectable compound to the nonstandard amino acid; and

(c) detecting the detectable compound in the subject,

thereby determining the distribution of the biofilm produced by the engineered bacterium in the subject.

58. A method for delivering a drug to a subject in need thereof, the method comprising:

(a) administering to the subject an engineered bacterium comprising a protein having at least one nonstandard amino acid, wherein said nonstandard amino acid comprises a first bioorthogonal reactive functional group;

(b) administering to the subject a drug comprising a second bioorthogonal reactive functional group, wherein the first bioorthogonal reactive functional group reacts with the second bioorthogonal reactive functional group in vivo, thereby attaching the drug to the nonstandard amino acid,

thereby delivering the drug to the subject in need thereof.

59. The method of any one of claims 54-58, wherein the engineered bacterium comprises:

(a) an orthogonal translation system comprising a heterologous gene encoding an orthogonal aminoacyl tR A synthetase and a heterologous gene encoding an orthogonal tRNA; and

(b) a heterologous nucleic acid encoding the protein having the at least one nonstandard amino acid; wherein the aminoacyl tRNA synthetase is capable of specifically aminoacylating the orthogonal tRNA with the nonstandard amino acid; and

wherein the orthogonal tRNA incorporates the nonstandard amino acid into the protein during translation.

60. The method of any one of claims 54-58, wherein step (a) is performed concurrently with step (b).

61. The method of any one of claims 54-58, wherein step (a) is performed before step (b).

62. The method of any one of claims 54-60, further comprising administering a nonstandard amino acid to the subject.

63. The method of claim 62, wherein the nonstandard amino acid is administered to the subject prior to administering the detectable compound or the drug to the subject.

64. The method of any one of claims 54-63, wherein the first bioorthogonal reactive functional group is selected from the group consisting of an azide moiety, a ketone moiety, an alkyne moiety, an alkene moiety, a tetrazine moiety, and a norbornene moiety.

65. The method of any one of claims 54-64, wherein the second bioorthogonal reactive functional group is selected from the group consisting of a hydrazine moiety, a

hydroxylamine moiety, an alkyne moiety, an azide moiety, a tetrazine moiety, and a norbornene moiety.

66. The method of any one of claims 54-65, wherein the first bioorthogonal reactive functional group is a ketone moiety, and the second bioorthogonal reactive functional group is either a hydrazine moiety or a hydroxylamine moiety.

67. The method of any one of claims 54-65, wherein the first bioorthogonal reactive functional group is an azide moiety, and the second bioorthogonal reactive functional group is an alkyne moiety.

68. The method of any one of claims 54-65, wherein the first bioorthogonal reactive functional group is an alkyne moiety, and the second bioorthpgonal reactive functional group is an azide moiety.

69. The method of any one of claims 54-68, wherein the first bioorthogonal reactive functional group reacts with the second bioorthogonal reactive functional group via a click chemistry reaction.

70. The method of any one of claims 54-69, wherein the reaction between the first bioorthogonal reactive functional group and the second bioorthogonal reactive functional group requires a catalytic agent.

71. The method of any one of claims 54-69, wherein the reaction between the first bioorthogonal reactive functional group and the second bioorthogonal reactive functional group does not require a catalytic agent.

72. The method of any one of claims 54-70 further comprising administering a catalytic agent to the subject.

73. The method of claim 70 or claims 72, wherein the catalytic agent is copper or nickel.

74. The method of any one of claims 54-73, wherein the detectable compound comprises a fluorescent moiety, a radioactive moiety, a colorimetric dye, a fluorescent dye, a luminescent dye, a magnetic resonance imaging (MRI) contrast agent, a CT contrast agent, a PET contrast agent, or an ultrasound contrast agent.

75. The method of any one of claims 54-74, wherein the protein further comprises a polypeptide linker.

76. The method of claim 75, wherein the polypeptide linker is flexible.

77. The method of any one of claims 54-76, wherein the protein further comprises a polypeptide tag.

78. The method of claim 77, wherein the polypeptide tag is selected from the group consisting of a poly-histidine tag, a myc tag, a FLAG tag, a hemagglutinin (HA) tag, and a V5 tag.

79. The method of any one of claims 54-78, wherein the protein is selected from the group consisting of a secreted protein, a membrane protein, or a bacterial extracellular matrix protein.

80. The method of claim 79, wherein the bacterial extracellular matrix protein is a curli fiber protein, a flagellar protein, or a pilin protein.

81. The method of any one of claims 54-79, wherein the protein is selected from the group consisting of InaV, Trat, and CsgA.

82. The method of claim 81, wherein the protein is CsgA.

83. The method of any one of claims 54-82, wherein the protein is CsgA and the nonstandard amino acid is located at an amino acid residue corresponding to residue 89 of wild-type CsgA.

84. The method of any one of claims 54-81, wherein the protein is InaV and the nonstandard amino acid is located at an amino acid residue corresponding to residue 174 of wild-type InaV.

85. The method of any one of claims 54-81, wherein the protein is Trat and the nonstandard amino acid is located at an amino acid residue corresponding to residue 200 of wild-type Trat.

86. The method of any one of claims 54-85, wherein the nonstandard amino acid is selected from the group consisting of -azidophenylalanine (p-AzF), azidohomoalanine (Aha), azidolysine, homopropargylglycine (Hpg), homoallylglycine (Hag), oxonorvaline (Onv), _jo-bromophenylalanine (p-BrF), /?-iodophenylalanine (p-IF), /?-ethynylphenylalanine

101 (p-EtF), /? ra-acetylphenylalanine (p-AcF), azidonorleucine (Anl), trans-crotylglycine (Teg), selenomethionine (Se-Met), 2-aminooctynoic acid (Aoa), and propargylglycine (Pra).

87. The method of claim 86, wherein the nonstandard amino acid is p-AzF.

88. The method of claim 87, wherein the detectable compound is DBCO-Cy5.

89. The method of any one of claims 54-88, wherein the engineered bacterium is nonpathogenic.

90. The method of any one of claims 54-89, wherein the engineered bacterium is of the species Escherichia coli.

91. The method of any one of claims 54-90, wherein the engineered bacterium is of an Escherichia coli strain selected from the group consisting of C321, C321.AA and

C321.AA.exp.

92. The method of any one of claims 54-90, wherein the engineered bacterium is of the Escherichia coli Nissle 1917 strain.

93. The method of any one of claims 54-92, wherein the engineered bacterium comprises a genome that has been recoded to reduce or eliminate a codon.

94. The method of any one of claims 54-93, wherein the engineered bacterium comprises a genome that has been recoded to reduce or eliminate a TAG codon.

95. The method of any one of claims 54-92, wherein the engineered bacterium comprises a genome that has not been recoded to reduce or eliminate a codon.

96. The method of any one of claims 54-95, wherein the engineered bacterium further comprises a prfA gene deletion.

102

97. The method of any one of claims 54-96, wherein the orthogonal aminoacyl tRNA synthetase is an Archea aminoacyl tRNA synthetase or a bacterial aminoacyl tRNA synthetase.

98. The method of any one of claims 54-97, wherein the heterologous gene encoding the orthogonal aminoacyl tRNA synthetase is a Methanosarcina, Desufitobacterium, Pyrococcus or Methanocaldococcus aminoacyl tRNA synthetase gene.

99. The method of any one of claims 54-98, wherein the heterologous gene encoding the orthogonal aminoacyl tRNA synthetase is a Methanosarcina mazei, Methanosarcina barkeri, Desulfitobacterium hafniense, Pyrococcus horikoshii, or Methanocaldococcus jannaschii aminoacyl tRNA synthetase gene.

100. The method of any one of claims 54-99, wherein the orthogonal translation system is located in the bacterial genome.

101. The method of any one of claims 54-99, wherein the orthogonal translation system is located in a plasmid.

102. The method of claim 101, wherein the plasmid is pEVOL-pAzF.

103. The method of any one of claims 54-102, wherein the expression of the heterologous nucleic acid is under the control of a constitutive promoter.

104. The method of any one of claims 54-102, wherein the expression of the heterologous nucleic acid is under the control of an inducible promoter.

105. The method of claim 104, wherein the inducible promoter is responsive to an inducer selected from the group consisting of IPTG, arabinose, and tetracycline.

106. The method of any one of claims 54-105, wherein the expression of the heterologous gene encoding the orthogonal aminoacyl tRNA synthetase and/or the orthogonal tRNA is under the control of a constitutive promoter.

103

107. The method of any one of claims 54-105, wherein the expression of the heterologous gene encoding the orthogonal aminoacyl tRNA synthetase and/or the orthogonal tRNA is under the control of an inducible promoter.

108. The method of claim 107, wherein the inducible promoter is responsive to an inducer selected from the group consisting of IPTG, arabinose, and tetracycline.

109. The method of claim 104, further comprising administering to the subject an inducer that regulates the inducible promoter.

1 10. The method of claim 107, further comprising administering to the subject an inducer that regulates the inducible promoter.

1 1 1. The method of claim 109 or claim 1 10, wherein the inducer is administered to the subject prior to administering the detectable compound or the drug to the subject.

112. The method of any one of claims 54-1 1 1 , wherein the engineered bacterium is administered to the subject orally or rectally.

1 13. The method of any one of claims 54-112, wherein the engineered bacterium is comprised in a pharmaceutical composition.

1 14. The method of any one of claims 54-1 12, wherein the detectable compound or the drug is administered to the subject orally, subcutaneously, intravenously, rectally, or transdermally.

1 15. The method of any one of claims 54-112, wherein the detectable compound or the drug is comprised in a pharmaceutical composition.

1 16. The method of any one of claims 109-1 15, wherein the inducer is comprised in a pharmaceutical composition.

104

1 17. The method of any one of claims 54-116, wherein the detectable compound or the drug further comprises a drug delivery vehicle selected from the group consisting of a nanocarrier, a nanoparticle, a liposome, a dendrimer, a carbon nanotube, a micelle, and a protein.

1 18. The method of claim 58, wherein the drug is delivered to the gastrointestinal tract of the subject.

1 19. The method of claim 58, wherein the drug is delivered to the mouth, esophagus, stomach, large intestine, small intestine, rectum, colon, or anal canal of the subject..

120. The method of claim 54, wherein the subject is a mouse.

121. The method of claim 120, wherein the detectable compound is Cy5-DBCO administered to the mouse at a concentration of 100 μΜ.

105