Genetic reprogramming of bacterial biofilms

Patent metadata

Field	Value
Publication number	US-9815871-B2
Application number	US-201414786304-A
Country	US
Kind code	B2
Filing date	Apr 23, 2014
Priority date	Apr 23, 2013
Publication date	Nov 14, 2017
Grant date	Nov 14, 2017

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 functionalized engineered CsgA polypeptide comprising an activity polypeptide fused to a full-length Escherichia coli CsgA polypeptide by a flexible linker sequence;
wherein the activity polypeptide comprises a conjugation domain;
wherein the conjugation domain is specifically bound to a partner conjugation domain of a target polypeptide;
wherein the target polypeptide comprises the partner conjugation domain fused to a functionalizing polypeptide;
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 (SEQ ID NO: 23);
wherein the partner conjugation domain is selected from the group consisting of SpyCatcher, streptavidin, and a peptide comprising aminoglycine;
and wherein the flexible linker sequence comprises at least 6 amino acids.

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

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

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

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

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

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

8. The functionalized engineered CsgA polypeptide of claim 1 , wherein the functionalizing polypeptide is an enzyme or an antibody.

9. The functionalized engineered CsgA polypeptide of claim 1 , wherein the conjugation domain or the functionalizing polypeptide further comprises an extracellular localization tag.

10. A biofilm including a functionalized engineered CsgA polypeptide comprising an activity polypeptide fused to a full length Escherichia coli CsgA polypeptide by a flexible linker sequence;
wherein the activity polypeptide comprises a conjugation domain;
wherein the conjugation domain is specifically bound to a partner conjugation domain of a target polypeptide;
wherein the target polypeptide comprises the partner conjugation domain fused to a functionalizing polypeptide;
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 (SEQ ID NO: 23);
wherein the partner conjugation domain is selected from the group consisting of SpyCatcher, streptavidin, and a peptide comprising aminoglycine;
and wherein the flexible linker sequence comprises at least 6 amino acids.

11. The functionalized engineered CsgA polypeptide of claim 1 , wherein the SpyTag comprises the amino acid sequence of SEQ ID NO: 9.

12. The biofilm of claim 10 , wherein the SpyTag comprises the amino acid sequence of SEQ ID NO: 9.

13. The biofilm of claim 10 , wherein the functionalizing polypeptide is an enzyme or an antibody.

14. A curli fiber comprising the functionalized engineered CsgA polypeptide of claim 1 .

15. A bacterial cell comprising the curli fiber of claim 14 .

Assignees

• Harvard College

Inventors

• Joshi Neel Satish
• Nguyen Peter Quoc
• Magarian Zsofia

Classifications

• A61P31/04

  Antibacterial agents · CPC title

• A61P43/00

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

• C07K2319/735

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

• C07K14/245 Primary

  Escherichia (G) · CPC title

• C07K2319/20

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