Msp nanopores and related methods
AssigneesThe UAB Research Foundation, University of Washington
InventorsJens H. GUNDLACH, Michael NIEDERWEIS, Thomas Z. BUTLER, Mikhail PAVLENOK, Mark A. TROLL
Life of this patent
Estimating the term…
Abstract
Provided herein are Mycobacterium smegmatis porin nanopores, systems that comprise these nanopores, and methods of using and making these nanopores. Such nanopores may be wild-type MspA porins, mutant MspA porins, wild-type MspA paralog porins, wild-type MspA homolog porins, mutant MspA paralog porins, mutant MspA homolog porins, or single-chain Msp porins. Also provided are bacterial strains capable of inducible Msp porin expression.
Claims59
A method comprising:
- applying an electric field to a Mycobacterium smegmatis porin (Msp) having a vestibule and a constriction zone that define a tunnel, wherein the Msp porin is positioned between a first conductive liquid medium and a second conductive liquid medium;
- and
- detecting an analyte present in the first or second conductive liquid media.
(canceled)
(canceled)
6-9. (canceled)
14-18. (canceled)
25-29. (canceled)
31-34. (canceled)
36-38. (canceled)
40-43. (canceled)
A method of modifying the conductance through the tunnel of a Mycobacterium smegmatis porin (Msp) comprising removing, adding, or replacing at least one amino acid in the vestibule or the constriction zone of a wild-type Msp.
A system comprising a Mycobacterium smegmatis porin (Msp) having a vestibule and a constriction zone that define a tunnel, wherein the tunnel is positioned between a first liquid medium and a second liquid medium, wherein at least one liquid medium comprises an analyte, and wherein the system is operative to detect a property of the analyte.
46-52. (canceled)
56-58. (canceled)
61-65. (canceled)
A mutant Mycobacterium smegmatis porin A (MspA) comprising:
- a vestibule and a constriction zone that define a tunnel, and
- at least a first mutant MspA monomer comprising a mutation at position 93 and a mutation at position 90, position 91, or both positions 90 and 91.
67-82. (canceled)
85-125. (canceled)
A nucleic acid sequence encoding a mutant single-chain Msp, wherein the nucleic acid sequence comprises:
- (a) a first and second nucleotide sequence, wherein the first nucleotide sequence encodes a first Msp monomer sequence and the second nucleotide sequence encodes a second Msp monomer sequence;
- and
- (b) a third nucleotide sequence encoding an amino acid linker sequence.
127-129. (canceled)
131-146. (canceled)
148-152. (canceled)
A nucleic acid sequence encoding a single-chain Msp, wherein the nucleic acid sequence comprises:
- (a) a first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequence, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences encode a first, second, third, fourth, fifth, sixth, seventh, and eighth Msp monomer sequence, respectively;
- and
- (b) a ninth nucleotide sequence encoding an amino acid linker sequence.
154-183. (canceled)
A mutant bacterial strain capable of inducible Msp monomer expression, the bacterial strain comprising:
- (a) a deletion of a wild-type MspA;
- (b) a deletion of a wild-type MspC;
- (c) a deletion of a wild-type MspD;
- (d) optionally a deletion of wild-type B;
- and
- (e) a vector comprising an inducible promoter operably linked to an Msp monomer nucleic acid sequence.
185-196. (canceled)
198-201. (canceled)
A mutant single-chain Msp encoded by a nucleic acid sequence comprising:
- a first and second nucleotide sequence, wherein the first nucleotide sequence encodes a first Msp monomer sequence and the second nucleotide sequence encodes a second Msp monomer sequence;
- and
- a third nucleotide sequence encoding an amino acid linker sequence.
A mutant single-chain Msp, optionally encoded by a nucleic acid sequence comprising:
- a first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequence, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences encode a first, second, third, fourth, fifth, sixth, seventh, and eighth Msp monomer sequence, respectively;
- and
- a ninth nucleotide sequence encoding an amino acid linker sequence.
Dependencies are inferred from the claim text.