Engineering the p7 Virporinfor Nanopore Sequencing

March 4, 2015

Max Genetti

Overview

• Nanopore Sequencing

• The p7 Pore

• Purification Techniques

• Lipid Membranes

Nanopore Sequencing

Nanopore Sequencing

Single Atom Sensitivity

calls

label C mC hmC fC caC

TGG 0.90 0.04 0.05 0.01 0.00

TCA 0.02 0.96 0.01 0.00 0.01

TCa 0.03 0.01 0.93 0.02 0.00

TTT 0.01 0.01 0.06 0.92 0.00

aaa 0.00 0.02 0.01 0.00 0.97

Need for New Pores

Pennisi 2012

α-Hemolysin MspA

~10 Nucleotides ~4 Nucleotides

Nonresolvable Sequences

Underrepresented kmers

•Bateriophage M13

•6407 nucleotides

The P7 Viroporin

Ca2+ Selectivity

Ca2+ Selectivity

Conformational Changes

Mutant

1 11 21 31 41 51 61

| | | | | | |

WTp7

JN1

GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA

GAKNVSVLSAASAAGSHGFFWGLTVVTSAWHVKGTLVPGATYLSLGVWPLLLVRLLRPHRALA

Amino Acid SubstitutionsR35TL28SL24TN16SN9SI6S

Expression System

Cleavage

HPLC Purification

Mass Spectrometry

WTp7

Mass SpectrometryJN1

SDS-PAGE

Lane1245679

SampleMolecular Weight StandardFirst Purification WTp7WTp7 Fraction 6WTp7 Fraction 7JN1 Fraction 6JN1 Fraction 7Molecular Weight Standard

Test on Nanopore Station

Conlusion