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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