Genetic Reprogramming of Bacterial Biofilms

Patent metadata

Field	Value
Publication number	US-2016185828-A1
Application number	US-201414786304-A
Country	US
Kind code	A1
Filing date	Apr 23, 2014
Priority date	Apr 23, 2013
Publication date	Jun 30, 2016
Grant date	—

Abstract

Described herein are methods and compositions relating to engineered curii fibers, e.g. CsgA polypeptide. In some embodiments, the methods and compositions described herein relate to functionalized biofilms. In one aspect, described herein is an engineered CsgA polypeptide, comprising a CsgA polypeptide with a C-terminal display tag flanking the CsgA polypeptide; wherein the display tag comprises an activity polypeptide and a linker sequence. In some embodiments, the display tag and/or the activity polypeptide comprises a polypeptide selected from the group consisting of metal binding domain (MBD); Spy Tag; graphene binding (GBP); carbon nanotube binding (CBP); gold binding (A3); CT43; FLAG; Z8; E14; QBP1; CLP12; and AFP8.

First claim

1 . An engineered CsgA polypeptide, comprising an activity polypeptide fused by a flexible linker sequence to a CsgA polypeptide; and
wherein the linker sequence comprises at least 6 amino acids.

2 . The engineered CsgA polypeptide of claim 1 , wherein the linker sequence consists of glycine and serine residues.

3 . The engineered CsgA polypeptide of claim 1 , wherein the activity polypeptide is selected from the group consisting of:
metal binding domain (MBD); SpyTag; graphene binding (GBP); carbon nanotube binding (CBP); gold binding (A3); CT43; FLAG; Z8; E14; QBP1; CLP12; and AFP8.

4 . The engineered CsgA polypeptide of claim 1 , wherein the activity polypeptide is a conjugation domain.

5 . The engineered CsgA polypeptide of claim 4 , wherein the conjugation domain is selected from the group consisting of:
SpyTag; biotin acceptor peptide (BAP); biotin carboxyl carrier protein (BCCP); and a peptide comprising a LPXTG motif.

6 .- 21 . (canceled)

22 . The engineered CsgA polypeptide of claim 1 wherein the flexible linker sequence includes from about 6 to about 100 amino acids.

23 . The engineered CsgA polypeptide of claim 1 wherein the flexible linker sequence includes from about 30 to about 100 amino acids.

24 . The engineered CsgA polypeptide of claim 1 wherein the flexible linker sequence includes from about 6 to about 30 amino acids.

25 . The engineered CsgA polypeptide of claim 1 wherein the flexible linker sequence includes from about 20 to about 50 amino acids.

26 . The engineered CsgA polypeptide of claim 1 wherein the flexible linker sequence includes from about 30 to about 50 amino acids.

27 . The engineered CsgA polypeptide of claim 1 wherein the activity polypeptide is an enzyme, a polypeptide that binds another molecule, a polypeptide that binds to tissue, a polypeptide that binds an inorganic nanoparticle, a polypeptide that binds a carbon-based nanostructure, a polypeptide that binds an environmental toxin, a polypeptide that binds to gold, a polypeptide that binds to silver, a polypeptide that binds to platinum, a polypeptide that binds to rhodium, a catalyst, an optically active polypeptide, an electrically conductive polypeptide, a polypeptide that binds to gold a conductive material, a polypeptide that binds to steel, a polypeptide that binds to glass, a metal ion binding protein, a binding domain, a ligand, an antibody or an epitope.

28 . The engineered CsgA polypeptide of claim 1 within a biofilm.

29 . The engineered CsgA polypeptide of claim 1 wherein the activity polypeptide is a conjugation domain and wherein a partner conjugation domain that is part of a target protein is bound to the conjugation domain.

30 . The engineered CsgA polypeptide of claim 1 wherein the activity polypeptide is a conjugation domain and wherein a partner conjugation domain that is part of a target protein is bound to the conjugation domain, wherein the target protein is an enzyme, a polypeptide that binds to another molecule, or an antibody.

31 . The engineered CsgA polypeptide of claim 1 wherein the activity polypeptide is a conjugation domain and wherein a partner conjugation domain that is part of a target protein is bound to the conjugation domain, and wherein the conjugation domain or the target protein includes an extracellular localization tag.

32 . An engineered CsgA polypeptide comprising an activity polypeptide fused by a flexible linker sequence to a CsgA polypeptide; and
wherein the linker sequence includes from about 30 to about 100 amino acids.

33 . The engineered CsgA polypeptide of claim 32 wherein the activity polypeptide is a conjugation domain, an enzyme, a polypeptide that binds another molecule, a polypeptide that binds to tissue, a polypeptide that binds an inorganic nanoparticle, a polypeptide that binds a carbon-based nanostructure, a polypeptide that binds an environmental toxin, a polypeptide that binds to gold, a polypeptide that binds to silver, a polypeptide that binds to platinum, a polypeptide that binds to rhodium, a catalyst, an optically active polypeptide, an electrically conductive polypeptide, a polypeptide that binds to gold a conductive material, a polypeptide that binds to steel, a polypeptide that binds to glass, a metal ion binding protein, a binding domain, a ligand, an antibody or an epitope.

34 . A biofilm including an engineered CsgA polypeptide comprising an activity polypeptide fused by a flexible linker sequence to a CsgA polypeptide;
wherein the linker sequence includes from about 30 to about 100 amino acids; and wherein the activity polypeptide is a conjugation domain, an enzyme, a polypeptide that binds another molecule, a polypeptide that binds to tissue, a polypeptide that binds an inorganic nanoparticle, a polypeptide that binds a carbon-based nanostructure, a polypeptide that binds an environmental toxin, a polypeptide that binds to gold, a polypeptide that binds to silver, a polypeptide that binds to platinum, a polypeptide that binds to rhodium, a catalyst, an optically active polypeptide, an electrically conductive polypeptide, a polypeptide that binds to gold a conductive material, a polypeptide that binds to steel, a polypeptide that binds to glass, a metal ion binding protein, a binding domain, a ligand, an antibody or an epitope.

Assignees

• Harvard College

Inventors

• Joshi Neel Satish
• Nguyen Peter Quoc
• Botyanszki Zsofia

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P31/04

  Antibacterial agents · CPC title

• C07K2319/735

  containing a domain for self-assembly, e.g. a viral coat protein (includes phage display) · CPC title

• C07K2319/20

  containing a tag with affinity for a non-protein ligand · CPC title

• C07K14/245 Primary

  Escherichia (G) · CPC title