Biosynthetic amyloid-based materials displaying functional protein sequences

Abstract

Embodiments of the present disclosure are directed to methods of genetically modifying bacteria to create amyloid-based materials, such as biofilms created by amyloid fibers, having nonnative functional polypeptides expressed thereon and connected thereto by a linker domain optimized for functioning of the non-native functional polypeptides. According to one aspect, the linker domain is optimized for functioning of a CsgA protein as is it assembled into an amyloid state and for functioning of the functional polypeptide. Exemplary biofilms may include living bacterial cells, non-living bacterial cells or combinations of living bacterial cells and non-living bacterial cells. Methods of making biofilms having non-native functional polypeptides attached thereto are provided.

1 . A method of making a biofilm comprising
 proliferating a bacteria cell including a nucleic acid sequence encoding a fusion of a CsgA protein linked to a non-native functional polypeptide by a linker to produce a population of bacteria cells expressing the fusion and forming a biofilm having the non-native functional polypeptide attached thereto,   wherein the linker is a polypeptide comprising 7 or more amino acids attached to either the C terminus or the N terminus of the CsgA protein.   
     
     
         2 . The method of  claim 1  wherein the nucleic acid sequence is introduced into the bacteria cell. 
     
     
         3 . The method of  claim 1  wherein the bacteria cell is  E. coli.    
     
     
         4 . The method of  claim 1  wherein the bacteria cell is a non-pathogenic bacteria. 
     
     
         5 . The method of  claim 1  wherein the bacteria cell is Nissle strain 1917 (EcN), MG1655, LSR10 or PHL628. 
     
     
         6 . The method of  claim 1  wherein the bacteria cell includes a genomic deletion of the CsgA gene. 
     
     
         7 . The method of  claim 1  wherein the bacteria cell has been genetically modified to remove a nucleic acid or nucleic acids encoding the CsgA protein. 
     
     
         8 . The method of  claim 1  wherein the bacteria cell has been genetically modified to remove the CsgA protein. 
     
     
         9 . The method of  claim 1  wherein the bacteria cell lacks expression of the natural CsgA gene is Nissle strain 1917 (EcN), MG1655, LSR10 or PHL628. 
     
     
         10 . The method of  claim 9  wherein the bacteria cell that lacks expression of the natural CsgA gene is Nissle strain 1917 (EcN), MG1655, LSR10 or PHL628. 
     
     
         11 . The method of  claim 1  wherein the bacteria cell is a knockout for the CsgA gene. 
     
     
         12 . The method of  claim 1  wherein the linker is between 7 and 250 amino acids. 
     
     
         13 . The method of  claim 1  wherein the linker is a flexible linker or a rigid linker. 
     
     
         14 . The method of  claim 1  wherein the linker is hydrophilic or hydrophobic. 
     
     
         15 . The method of  claim 1  wherein the linker comprises a repeating amino acid subunit. 
     
     
         16 . The method of  claim 1  wherein the linker comprises two repeating amino acid subunits or more. 
     
     
         17 . The method of  claim 1  wherein the linker comprises 3 repeating amino acid subunits or more. 
     
     
         18 . The method of  claim 1  wherein the linker comprises [GGGS] n  wherein n is an integer from 2 to 20 (SEQ ID NO:12). 
     
     
         19 . The method of  claim 1  wherein the linker comprises [GGGS] n  wherein n is an integer being 3, 6, or 12 (SEQ ID NO:5-7). 
     
     
         20 . The method of  claim 1  wherein the linker comprises [P] n  wherein n is an integer from 1 to 30 (SEQ ID NO:13). 
     
     
         21 . The method of  claim 1  wherein the linker comprises [P] n  wherein n is an integer being 12 or 24 (SEQ ID NO:8-9). 
     
     
         22 . The method of  claim 1  wherein the linker comprises [EAAAK] n  wherein n is an integer from 1 to 15 (SEQ ID NO:15). 
     
     
         23 . The method of  claim 1  wherein the linker comprises [EAAAK] n  wherein n is an integer being 3 or 9 (SEQ ID NO:10-11). 
     
     
         24 . The method of  claim 1  wherein the linker comprises one or more of glycine, serine, alanine or leucine. 
     
     
         25 . The method of  claim 1  wherein the linker comprises one or more of [GGGS] n  wherein n is an integer from 2 to 20 (SEQ ID NO:12), [P] n  wherein n is an integer from 1 to 30 (SEQ ID NO:13) or [EAAAK] n  wherein n is an integer from 1 to 15 (SEQ ID NO:15). 
     
     
         26 . The method of  claim 1  wherein the linker is cleavable. 
     
     
         27 . The method of  claim 1  wherein the linker is cleavable by an enzyme. 
     
     
         28 . The method of  claim 1  wherein the non-native functional polypeptide is releasable. 
     
     
         29 . The method of  claim 1  wherein the linker is GGGSGGGSGGGS, GGGSGGGSGGGSGGGSGGGSGGGS, GGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 5-7), PPPPPPPPPPPP, PPPPPPPPPPPPPPPPPPPPPPPP (SEQ ID NO:8-9), EAAAKEAAAKEAAAK, or EAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAK (SEQ ID NO:10-11). 
     
     
         30 . The method of  claim 1  wherein the non-native functional polypeptide is a therapeutic polypeptide, a diagnostic polypeptide, a tissue-binding polypeptide, a cell-binding polypeptide, an antimicrobial polypeptide, an anticancer polypeptide, an anti-inflammatory polypeptide, a polymer binding polypeptide, a metabolite binding polypeptide, a targeting polypeptide or a polypeptide that is a first pair of a binding pair of molecules. 
     
     
         31 . The method of  claim 1  wherein the bacteria cell is Nissle strain 1917 (EcN) and the linker is [GGGS] n  wherein n is and integer being 3, 6, or 12 (SEQ ID NO:5-7). 
     
     
         32 . A non-naturally occurring fusion of a CsgA protein linked to a non-native functional polypeptide by a linker, wherein the linker comprises [GGGS], wherein n is an integer from 2 to 20 (SEQ ID NO:12). 
     
     
         33 . A nucleic acid sequence encoding a fusion of a CsgA protein linked to a non-native functional polypeptide by a linker, wherein the linker comprises [GGGS] n  wherein n is an integer from 2 to 20 (SEQ ID NO:12). 
     
     
         34 . A vector comprising a nucleic acid sequence encoding a fusion of a CsgA protein linked to a non-native functional polypeptide by a linker, wherein the linker comprises [GGGS] n  wherein n is an integer from 2 to 20 (SEQ ID NO:12). 
     
     
         35 . A bacteria cell including a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a non-native functional polypeptide by a linker, wherein the linker comprises [GGGS] n  wherein n is an integer from 2 to 20 (SEQ ID NO:12). 
     
     
         36 . A bacteria cell including a vector comprising a nucleic acid sequence encoding a fusion of a CsgA protein linked to a non-native functional polypeptide by a linker, wherein the linker comprises [GGGS] n  wherein n is an integer from 2 to 20 (SEQ ID NO:12). 
     
     
         37 . A bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a non-native functional polypeptide by a linker, wherein the linker comprises [GGGS] n  wherein n is an integer from 2 to 20 (SEQ ID NO:12). 
     
     
         38 . A biofilm including a bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a non-native functional polypeptide by a linker, wherein the linker comprises [GGGS] n  wherein n is an integer from 2 to 20 (SEQ ID NO:12). 
     
     
         39 . A method of delivering a bacteria cell to a tissue within an organism comprising
 introducing into the organism a bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a tissue binding polypeptide by a linker, wherein the linker comprises [GGGS] n  wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the bacteria cell attaches to the tissue by the tissue binding polypeptide thereby localizing the bacteria cell to the tissue.   
     
     
         40 . The method of  claim 39  wherein the bacteria cell proliferates and a biofilm including a population of bacteria cells is formed that is attached to the tissue. 
     
     
         41 . The method of  claim 39  wherein the bacteria cell is Nissle strain 1917 (EcN). 
     
     
         42 . The method of  claim 39  wherein the tissue binding polypeptide is CP15, P8, A1, T18, TTF1, TTF2 or TTF3. 
     
     
         43 . The method of  claim 39  wherein the tissue within the organism is gastrointestinal tract epithelial tissue. 
     
     
         44 . The method of  claim 39  wherein the tissue within the organism is Peyer's Patches. 
     
     
         45 . The method of  claim 39  wherein the tissue within the organism is an infection site. 
     
     
         46 . The method of  claim 39  wherein the tissue within the organism is an injured area of epithelium. 
     
     
         47 . The method of  claim 39  wherein the tissue within the organism is tumor tissue. 
     
     
         48 . The method of  claim 39  wherein the tissue within the organism is a site of inflammation. 
     
     
         49 . The method of  claim 39  wherein the tissue within the organism includes colon carcinoma cells. 
     
     
         50 . The method of  claim 39  wherein the tissue within the organism is gut mucosa. 
     
     
         51 . The method of  claim 39  wherein the tissue within the organism includes M cells. 
     
     
         52 . A method of treating an organism with inflammation of the gastrointestinal tract comprising
 introducing into the organism a Nissle strain 1917 (EcN) bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a tissue binding polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the Nissle strain 1917 (EcN) bacteria cell attaches to tissue of the gastrointestinal tract by the tissue binding polypeptide thereby localizing the Nissle strain 1917 (EcN) bacteria cell to the tissue in a manner to reduce the inflammation.   
     
     
         53 . The method of  claim 52  wherein the Nissle strain 1917 (EcN) bacteria cell proliferates and a biofilm including a population of Nissle strain 1917 (EcN) bacteria cells is formed that is attached to the tissue. 
     
     
         54 . The method of  claim 52  wherein the tissue binding polypeptide is CP15, P8, A1, T18, TTF1, TTF2 or TTF3. 
     
     
         55 . The method of  claim 52  wherein the inflammation results from inflammatory bowel disease. 
     
     
         56 . The method of  claim 52  wherein the inflammation results from Crohn's disease or ulcerative colitis. 
     
     
         57 . A method of treating an organism with inflammation of the gastrointestinal tract comprising
 introducing into the organism a Nissle strain 1917 (EcN) bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to an anti-inflammatory polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the Nissle strain 1917 (EcN) bacteria cell proliferates within the gastrointestinal tract and a biofilm including a population of Nissle strain 1917 (EcN) bacteria cells is formed that includes the anti-inflammatory polypeptide in a manner to reduce the inflammation.   
     
     
         58 . The method of  claim 57  wherein the anti-inflammatory polypeptide is TTF1, TTF2 or TTF3. 
     
     
         59 . The method of  claim 57  wherein the inflammation results from inflammatory bowel disease or Crohn's disease. 
     
     
         60 . The method of  claim 57  wherein the anti-inflammatory polypeptide is released. 
     
     
         61 . The method of  claim 57  wherein the linker is a cleavable linker and the anti-inflammatory polypeptide is released by cleaving the linker. 
     
     
         62 . The method of  claim 57  wherein the linker is a cleavable linker and the anti-inflammatory polypeptide is released by cleaving the linker with an enzyme. 
     
     
         63 . The method of  claim 57  wherein the linker is a cleavable linker and the anti-inflammatory polypeptide is released by cleaving the linker with a protease produced as a result of the inflammation. 
     
     
         64 . The method of  claim 57  wherein the linker is a cleavable linker and the anti-inflammatory polypeptide is released by cleaving the linker with an MMP protease. 
     
     
         65 . A method of treating an organism with cancer of the gastrointestinal tract comprising
 introducing into the organism a Nissle strain 1917 (EcN) bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to an anti-cancer polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the Nissle strain 1917 (EcN) bacteria cell proliferates within the gastrointestinal tract and a biofilm including a population of Nissle strain 1917 (EcN) bacteria cells is formed that includes the anti-cancer polypeptide in a manner to reduce proliferation of the cancer.   
     
     
         66 . The method of  claim 65  wherein the anti-cancer polypeptide is a growth inhibiting biologic. 
     
     
         67 . The method of  claim 65  wherein the anti-cancer polypeptide is released. 
     
     
         68 . The method of  claim 65  wherein the linker is a cleavable linker and the anti-cancer polypeptide is released by cleaving the linker. 
     
     
         69 . The method of  claim 65  wherein the linker is a cleavable linker and the anti-cancer polypeptide is released by cleaving the linker with an enzyme. 
     
     
         70 . The method of  claim 65  wherein the linker is a cleavable linker and the anti-cancer polypeptide is released by cleaving the linker with an enzyme within the gastrointestinal tract. 
     
     
         71 . A method of treating an organism with microbial pathogens in the gastrointestinal tract comprising
 introducing into the organism a Nissle strain 1917 (EcN) bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to an anti-microbial polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the Nissle strain 1917 (EcN) bacteria cell proliferates within the gastrointestinal tract and a biofilm including a population of Nissle strain 1917 (EcN) bacteria cells is formed that includes the anti-microbial polypeptide in a manner to reduce proliferation of the microbial pathogens.   
     
     
         72 . The method of  claim 71  wherein the microbial pathogen is  Clostridium difficile.    
     
     
         73 . The method of  claim 71  wherein the anti-microbial polypeptide is coprisin, thurcin CD, a lanibiotic, nisin, actagardine, a cathelicidin or LL-37. 
     
     
         74 . The method of  claim 71  wherein the anti-microbial polypeptide is released. 
     
     
         75 . The method of  claim 71  wherein the linker is a cleavable linker and the anti-microbial polypeptide is released by cleaving the linker. 
     
     
         76 . The method of  claim 71  wherein the linker is a cleavable linker and the anti-microbial polypeptide is released by cleaving the linker with an enzyme. 
     
     
         77 . The method of  claim 71  wherein the linker is a cleavable linker and the anti-microbial polypeptide is released by cleaving the linker with an enzyme within the gastrointestinal tract. 
     
     
         78 . The method of  claim 71  wherein the linker is a cleavable linker and the anti-microbial polypeptide is released by cleaving the linker with protease CD2830 within the gastrointestinal tract. 
     
     
         79 . The method of  claim 71  wherein the linker is a cleavable linker and the anti-microbial polypeptide is released when exposed to microbial pathogen. 
     
     
         80 . A method of delivering a diagnostic polypeptide to the gastrointestinal tract of an organism comprising
 introducing into the organism a Nissle strain 1917 (EcN) bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a diagnostic polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the Nissle strain 1917 (EcN) bacteria cell proliferates within the gastrointestinal tract and a biofilm including a population of Nissle strain 1917 (EcN) bacteria cells is formed that includes the diagnostic polypeptide.   
     
     
         81 . The method of  claim 80  wherein the diagnostic polypeptide is detected. 
     
     
         82 . The method of  claim 80  wherein the diagnostic polypeptide further comprises an imaging agent or a dye. 
     
     
         83 . The method of  claim 80  wherein the diagnostic polypeptide is released. 
     
     
         84 . The method of  claim 80  wherein the linker is a cleavable linker and the diagnostic polypeptide is released by cleaving the linker. 
     
     
         85 . The method of  claim 80  wherein the linker is a cleavable linker and the diagnostic polypeptide is released by cleaving the linker with an enzyme. 
     
     
         86 . The method of  claim 80  wherein the linker is a cleavable linker and the diagnostic polypeptide is released by cleaving the linker with an enzyme within the gastrointestinal tract. 
     
     
         87 . A method of binding capture targets within the gastrointestinal tract of an organism comprising
 introducing into the organism a Nissle strain 1917 (EcN) bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a capture agent polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the Nissle strain 1917 (EcN) bacteria cell proliferates within the gastrointestinal tract and a biofilm including a population of Nissle strain 1917 (EcN) bacteria cells is formed that includes the capture agent polypeptide, and   wherein the capture target binds to the capture agent polypeptide.   
     
     
         88 . The method of  claim 87  wherein the biofilm including bound capture target is excreted from the organism. 
     
     
         89 . The method of  claim 87  wherein the biofilm including bound capture target is removed from tissue and is excreted from the organism. 
     
     
         90 . The method of  claim 87  wherein the biofilm including bound capture target is removed from tissue by natural processes and is excreted from the organism. 
     
     
         91 . The method of  claim 87  wherein the capture agent polypeptide is released. 
     
     
         92 . The method of  claim 87  wherein the linker is a cleavable linker and the capture agent polypeptide is released by cleaving the linker. 
     
     
         93 . The method of  claim 87  wherein the linker is a cleavable linker and the capture agent polypeptide is released by cleaving the linker with an enzyme. 
     
     
         94 . The method of  claim 87  wherein the linker is a cleavable linker and the capture agent polypeptide is released by cleaving the linker with an enzyme within the gastrointestinal tract. 
     
     
         95 . A method of delivering a functional agent to the gastrointestinal tract of an organism comprising
 introducing into the organism a Nissle strain 1917 (EcN) bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a polypeptide that is a first member of a binding pair of molecules by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the Nissle strain 1917 (EcN) bacteria cell proliferates within the gastrointestinal tract and a biofilm including a population of Nissle strain 1917 (EcN) bacteria cells is formed that includes the first member of a binding pair of molecules,   introducing into the gastrointestinal tract a second member of the binding pair of molecules having a functional agent attached thereto,   wherein the first and second members of the binding pair of molecules bind to each other, thereby attaching the functional agent to the biofilm.   
     
     
         96 . A bacteria cell genetically modified to lack a native sequence encoding a CsgA protein. 
     
     
         97 . The bacteria of  claim 96  which is  E. coli.    
     
     
         98 . The bacteria of  claim 96  which is Nissle strain 1917 (EcN). 
     
     
         99 . A bacteria cell genetically modified to lack a native sequence encoding a CsgA protein and including a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a non-native functional polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12). 
     
     
         100 . A bacteria cell genetically modified to lack a native sequence encoding a CsgA protein and including a vector comprising a nucleic acid sequence encoding a fusion of a CsgA protein linked to a non-native functional polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12). 
     
     
         101 . A bacteria cell genetically modified to lack a native sequence encoding a CsgA protein and expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a non-native functional polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12). 
     
     
         102 . A method of treating an organism with inflammation of the gastrointestinal tract comprising
 introducing into the organism an  E. coli  bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a tissue binding polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the  E. coli  bacteria cell attaches to tissue of the gastrointestinal tract by the tissue binding polypeptide thereby localizing the  E. coli  bacteria cell to the tissue in a manner to reduce the inflammation.   
     
     
         103 . The method of  claim 102  wherein the  E. coli  bacteria cell proliferates and a biofilm including a population of  E. coli  bacteria cells is formed that is attached to the tissue. 
     
     
         104 . The method of  claim 102  wherein the tissue binding polypeptide is CP15, P8, A1, T18, TTF1, TTF2 or TTF3. 
     
     
         105 . The method of  claim 102  wherein the inflammation results from inflammatory bowel disease. 
     
     
         106 . The method of  claim 102  wherein the inflammation results from Crohn's disease or ulcerative colitis. 
     
     
         107 . A method of treating an organism with inflammation of the gastrointestinal tract comprising
 introducing into the organism an  E. coli  bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to an anti-inflammatory polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the  E. coli  bacteria cell proliferates within the gastrointestinal tract and a biofilm including a population of  E. coli  bacteria cells is formed that includes the anti-inflammatory polypeptide in a manner to reduce the inflammation.   
     
     
         108 . The method of  claim 107  wherein the anti-inflammatory polypeptide is TTF1, TTF2 or TTF3. 
     
     
         109 . The method of  claim 107  wherein the inflammation results from inflammatory bowel disease or Crohn's disease. 
     
     
         110 . The method of  claim 107  wherein the anti-inflammatory polypeptide is released. 
     
     
         111 . The method of  claim 107  wherein the linker is a cleavable linker and the anti-inflammatory polypeptide is released by cleaving the linker. 
     
     
         112 . The method of  claim 107  wherein the linker is a cleavable linker and the anti-inflammatory polypeptide is released by cleaving the linker with an enzyme. 
     
     
         113 . The method of  claim 107  wherein the linker is a cleavable linker and the anti-inflammatory polypeptide is released by cleaving the linker with a protease produced as a result of the inflammation. 
     
     
         114 . The method of  claim 107  wherein the linker is a cleavable linker and the anti-inflammatory polypeptide is released by cleaving the linker with an MMP protease. 
     
     
         115 . A method of treating an organism with cancer of the gastrointestinal tract comprising
 introducing into the organism an  E. coli  bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to an anti-cancer polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the  E. coli  bacteria cell proliferates within the gastrointestinal tract and a biofilm including a population of  E. coli  bacteria cells is formed that includes the anti-cancer polypeptide in a manner to reduce proliferation of the cancer.   
     
     
         116 . The method of  claim 115  wherein the anti-cancer polypeptide is a growth inhibiting biologic. 
     
     
         117 . The method of  claim 115  wherein the anti-cancer polypeptide is released. 
     
     
         118 . The method of  claim 115  wherein the linker is a cleavable linker and the anti-cancer polypeptide is released by cleaving the linker. 
     
     
         119 . The method of  claim 115  wherein the linker is a cleavable linker and the anti-cancer polypeptide is released by cleaving the linker with an enzyme. 
     
     
         120 . The method of  claim 115  wherein the linker is a cleavable linker and the anti-cancer polypeptide is released by cleaving the linker with an enzyme within the gastrointestinal tract. 
     
     
         121 . A method of treating an organism with microbial pathogens in the gastrointestinal tract comprising
 introducing into the organism an  E. coli  bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to an anti-microbial polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the  E. coli  bacteria cell proliferates within the gastrointestinal tract and a biofilm including a population of  E. coli  bacteria cells is formed that includes the anti-microbial polypeptide in a manner to reduce proliferation of the microbial pathogens.   
     
     
         122 . The method of  claim 121  wherein the microbial pathogen is  Clostridium difficile.    
     
     
         123 . The method of  claim 121  wherein the anti-microbial polypeptide is coprisin, thurcin CD, a lanibiotic, nisin, actagardine, a cathelicidin or LL-37. 
     
     
         124 . The method of  claim 121  wherein the anti-microbial polypeptide is released. 
     
     
         125 . The method of  claim 121  wherein the linker is a cleavable linker and the anti-microbial polypeptide is released by cleaving the linker. 
     
     
         126 . The method of  claim 121  wherein the linker is a cleavable linker and the anti-microbial polypeptide is released by cleaving the linker with an enzyme. 
     
     
         127 . The method of  claim 121  wherein the linker is a cleavable linker and the anti-microbial polypeptide is released by cleaving the linker with an enzyme within the gastrointestinal tract. 
     
     
         128 . The method of  claim 121  wherein the linker is a cleavable linker and the anti-microbial polypeptide is released by cleaving the linker with protease CD2830 within the gastrointestinal tract. 
     
     
         129 . The method of  claim 121  wherein the linker is a cleavable linker and the anti-microbial polypeptide is released when exposed to microbial pathogen. 
     
     
         130 . A method of delivering a diagnostic polypeptide to the gastrointestinal tract of an organism comprising
 introducing into the organism an  E. coli  bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a diagnostic polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the  E. coli  bacteria cell proliferates within the gastrointestinal tract and a biofilm including a population of  E. coli  bacteria cells is formed that includes the diagnostic polypeptide.   
     
     
         131 . The method of  claim 130  wherein the diagnostic polypeptide is detected. 
     
     
         132 . The method of  claim 130  wherein the diagnostic polypeptide further comprises an imaging agent or a dye. 
     
     
         133 . The method of  claim 130  wherein the diagnostic polypeptide is released. 
     
     
         134 . The method of  claim 130  wherein the linker is a cleavable linker and the diagnostic polypeptide is released by cleaving the linker. 
     
     
         135 . The method of  claim 130  wherein the linker is a cleavable linker and the diagnostic polypeptide is released by cleaving the linker with an enzyme. 
     
     
         136 . The method of  claim 130  wherein the linker is a cleavable linker and the diagnostic polypeptide is released by cleaving the linker with an enzyme within the gastrointestinal tract. 
     
     
         137 . A method of binding capture targets within the gastrointestinal tract of an organism comprising
 introducing into the organism an  E. coli  bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a capture agent polypeptide by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the  E. coli  bacteria cell proliferates within the gastrointestinal tract and a biofilm including a population of  E. coli  bacteria cells is formed that includes the capture agent polypeptide, and   wherein the capture target binds to the capture agent polypeptide.   
     
     
         138 . The method of  claim 137  wherein the biofilm including bound capture target is excreted from the organism. 
     
     
         139 . The method of  claim 137  wherein the biofilm including bound capture target is removed from tissue and is excreted from the organism. 
     
     
         140 . The method of  claim 137  wherein the biofilm including bound capture target is removed from tissue by natural processes and is excreted from the organism. 
     
     
         141 . The method of  claim 137  wherein the capture agent polypeptide is released. 
     
     
         142 . The method of  claim 137  wherein the linker is a cleavable linker and the capture agent polypeptide is released by cleaving the linker. 
     
     
         143 . The method of  claim 137  wherein the linker is a cleavable linker and the capture agent polypeptide is released by cleaving the linker with an enzyme. 
     
     
         144 . The method of  claim 137  wherein the linker is a cleavable linker and the capture agent polypeptide is released by cleaving the linker with an enzyme within the gastrointestinal tract. 
     
     
         145 . A method of delivering a functional agent to the gastrointestinal tract of an organism comprising
 introducing into the organism an  E. coli  bacteria cell expressing a foreign nucleic acid sequence encoding a fusion of a CsgA protein linked to a polypeptide that is a first member of a binding pair of molecules by a linker, wherein the linker comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);   wherein the  E. coli  bacteria cell proliferates within the gastrointestinal tract and a biofilm including a population of  E. coli  bacteria cells is formed that includes the first member of a binding pair of molecules,   introducing into the gastrointestinal tract a second member of the binding pair of molecules having a functional agent attached thereto,   wherein the first and second members of the binding pair of molecules bind to each other, thereby attaching the functional agent to the biofilm.