Methods and compositions for the detection of target molecules

Abstract

The present disclosure provides methods and compositions for the detection of target molecules, comprising modified bacteriophages engineered to express a luciferase.

1 . A method for detecting a target molecule using a protein VII (pVII)-modified bacteriophage, comprising:
 a) contacting an immobilized target molecule with a bacteriophage encoding a luciferase and comprising a target-molecule specific insert in pVII, wherein the bacteriophage specifically binds the target molecule, under conditions that promote binding of the bacteriophage to the target molecule, to produce a target molecule-bacteriophage complex;   b) contacting the intact target molecule-bacteriophage complex with a bacterial strain susceptible to infection by the bacteriophage;   c) incubating the target molecule-bacteriophage complex with the bacterial strain under conditions that promote the propagation of the bacteriophage in the bacteria and expression of the luciferase, to produce a bacteriophage-infected bacterial culture comprising luciferase;   d) adding to the bacteriophage-infected bacterial culture a substrate for the luciferase; and   e) measuring the presence and/or magnitude of fluorescence produced from the action of the luciferase on its substrate, thereby detecting the presence of the target molecule.   
     
     
         2 . The method of  claim 1 , wherein the target molecule comprises a first affinity tag and the bacteriophage comprises a second affinity tag, wherein the first and second affinity tags specifically bind. 
     
     
         3 . The method of  claim 2 , wherein the first and second affinity tags are enzymatically or chemically coupled to the target molecule and the bacteriophage. 
     
     
         4 . The method of  claim 2 , wherein the first affinity tag is streptavidin and the second affinity tag is biotin. 
     
     
         5 . The method of  claim 1 , wherein the bacteriophage is engineered to bind directly to the target molecule using phage display. 
     
     
         6 . The method of  claim 1 , wherein the bacteriophage is derived from an M13, T, T7, or λ bacteriophage. 
     
     
         7 . The method of  claim 1 , wherein the bacteriophage comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. 
     
     
         8 . A method for detecting a target molecule using a protein VII (pVII)-modified bacteriophage, comprising:
 a) contacting an immobilized target molecule with a first affinity ligand that specifically binds the target molecule, under conditions that promote target molecule-affinity ligand binding, to form a target molecule-first affinity ligand complex, and optionally contacting the target molecule-first affinity ligand complex with one or more additional affinity ligands under conditions that promote affinity ligand-affinity ligand binding, to produce a target molecule-first affinity ligand-additional affinity ligand complex, wherein the one or more affinity ligands are added sequentially, and wherein each successive affinity ligand specifically binds to the affinity ligand added immediately previous;   b) contacting the target molecule-first affinity ligand complex or the target molecule-first affinity ligand-additional affinity ligand complex with a bacteriophage encoding a luciferase and comprising an affinity ligand-specific insert at pVII, wherein the bacteriophage binds to the first affinity ligand, or if one or more additional affinity ligands are used, binds to the ultimate affinity ligand, to form a target molecule-affinity ligand-bacteriophage complex;   c) contacting the intact target molecule-affinity ligand-bacteriophage complex with a bacterial strain susceptible to infection by the bacteriophage;   d) incubating the target molecule-affinity ligand-bacteriophage complex with the bacterial strain under conditions that promote the propagation of the bacteriophage in the bacteria and expression of the luciferase, to produce a bacteriophage-infected bacterial culture comprising luciferase;   e) adding to the bacteriophage-infected bacterial culture a substrate for the luciferase; and   f) measuring the presence and/or magnitude of fluorescence produced from the action the luciferase on its substrate, thereby detecting the presence of the target molecule.   
     
     
         9 . The method of  claim 8 , wherein the first affinity ligand, or if one or more additional affinity ligands are used, the ultimate affinity ligand, comprises a first affinity tag, and the bacteriophage comprises a second affinity tag, wherein the first and second affinity tags specifically bind. 
     
     
         10 . The method of  claim 8 , wherein the first and second affinity tags are enzymatically or chemically coupled to the affinity ligand and the bacteriophage. 
     
     
         11 . The method of  claim 8 , wherein the first affinity tag is streptavidin and the second affinity tag is biotin. 
     
     
         12 . The method of  claim 8 , wherein the bacteriophage is engineered to bind directly to the affinity ligand using phage display. 
     
     
         13 . The method of  claim 8 , wherein the bacteriophage is derived from an M13, T, 7, or λ bacteriophage. 
     
     
         14 . The method of  claim 8 , wherein the bacteriophage comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. 
     
     
         15 . A protein VII (pVII)-modified bacteriophage comprising a nucleic acid encoding a luciferase, wherein the bacteriophage comprises a target-specific insert at protein VII (pVII). 
     
     
         16 . The bacteriophage, wherein the bacteriophage is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO: 4. 
     
     
         17 . The bacteriophage of  claim 15 , wherein the bacteriophage comprises an affinity tag. 
     
     
         18 . The bacteriophage nucleic acid of  claim 17 , wherein the affinity tag is enzymatically or chemically coupled to the bacteriophage. 
     
     
         19 . The bacteriophage nucleic acid of  claim 15 , wherein the bacteriophage is engineered to bind directly to an affinity ligand or to a target molecule using phage display.