This document discusses engineering the p7 viroporin for use in nanopore sequencing. It describes how the p7 pore was mutated (JN1 variant) to increase its stability and selectivity for calcium ions. Purification techniques such as HPLC and SDS-PAGE were used to isolate the mutated JN1 pore from other proteins. Finally, initial tests on a nanopore sequencing station showed that the mutated JN1 pore could be used to distinguish nucleotide sequences in a strand of DNA or RNA.

1. Engineering the p7 Virporin
for Nanopore Sequencing
March 4, 2015
Max Genetti

2. Overview
• Nanopore Sequencing
• The p7 Pore
• Purification Techniques
• Lipid Membranes

3. Nanopore Sequencing

4. Nanopore Sequencing

5. 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

6. Need for New Pores
Pennisi 2012
α-Hemolysin MspA
~10 Nucleotides ~4 Nucleotides

7. Nonresolvable Sequences

8. Underrepresented kmers
•Bateriophage M13
•6407 nucleotides

9. The P7 Viroporin

10. Ca2+ Selectivity

11. Ca2+ Selectivity

12. Conformational Changes

13. Mutant
1 11 21 31 41 51 61
| | | | | | |
WTp7
JN1
GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA
GAKNVSVLSAASAAGSHGFFWGLTVVTSAWHVKGTLVPGATYLSLGVWPLLLVRLLRPHRALA
Amino Acid Substitutions
R35T
L28S
L24T
N16S
N9S
I6S

14. Expression System

15. Cleavage

16. HPLC Purification

17. Mass Spectrometry
WTp7

18. Mass Spectrometry
JN1

19. SDS-PAGE
Lane
1
2
4
5
6
7
9
Sample
Molecular Weight Standard
First Purification WTp7
WTp7 Fraction 6
WTp7 Fraction 7
JN1 Fraction 6
JN1 Fraction 7
Molecular Weight Standard

20. Test on Nanopore Station

21. Conlusion