Genetic reprogramming of bacterial biofilms

Patent metadata

Field	Value
Publication number	US-10550160-B2
Application number	US-201715808237-A
Country	US
Kind code	B2
Filing date	Nov 9, 2017
Priority date	Apr 23, 2013
Publication date	Feb 4, 2020
Grant date	Feb 4, 2020

Abstract

Described herein are methods and compositions relating to engineered curli fibers, e.g. CsgA polypeptide. In some embodiments, the methods and compositions described herein relate to functionalized biofilms.

First claim

What is claimed herein is:

1. A method of displaying an activity polypeptide on the surface of a biofilm, the method comprising:
expressing a functionalized engineered CsgA polypeptide on a microbial cell, wherein the functionalized engineered CsgA polypeptide has a C-terminal display tag comprising the activity polypeptide fused to an Escherichia coli CsgA polypeptide by a flexible linker sequence, wherein the activity polypeptide comprises a binding conjugation domain having specific binding affinity for a partner interacting domain; and
exposing the functionalized engineered CsgA polypeptide to a target protein, wherein the target protein is fused to the partner interacting domain, wherein the binding conjugation domain of the activity polypeptide specifically binds to the partner interacting domain fused to the target protein,
thereby displaying the activity polypeptide on the surface of the biofilm.

2. The method of claim 1 , wherein the binding 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 (SEQ ID NO: 23).

3. The method of claim 1 , wherein the partner interacting domain is selected from the group consisting of SpyCatcher, streptavidin, and a peptide comprising aminoglycine.

4. The method of claim 1 , wherein the flexible linker sequence comprises at least 6 amino acids.

5. The method of claim 1 , wherein the flexible linker sequence consists of glycine and serine residues.

6. The method of claim 1 , wherein the flexible linker sequence includes from 6 to 100 amino acids.

7. The method of claim 1 , wherein the flexible linker sequence includes from 30 to 100 amino acids.

8. The method of claim 1 , wherein the flexible linker sequence includes from 6 to 30 amino acids.

9. The method of claim 1 , wherein the flexible linker sequence includes from 20 to 50 amino acids.

10. The method of claim 1 , wherein the flexible linker sequence includes from 30 to 50 amino acids.

11. The method of claim 1 , wherein the target protein is a fluorescent protein.

12. The method of claim 1 , wherein the binding conjugation domain further comprises an extracellular localization tag.

13. The method of claim 1 , wherein the SpyTag comprises the amino acid sequence of SEQ ID NO: 9.

14. The method of claim 1 , wherein the CsgA polypeptide is a full-length Escherichia coli CsgA polypeptide.

Assignees

• Harvard College

Inventors

• Joshi Neel Satish
• Nguyen Peter Q
• Magarian 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

• C07K14/245 Primary

  Escherichia (G) · CPC title

• C07K2319/735

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

• C07K14/21

  from Pseudomonadaceae (F) · CPC title