Battling Infective Endocarditis with Reverse Vaccinology: the molecular approach

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Battling Infective Endocarditis with Reverse
Vaccinology the molecular approach

• Katie Vollmer
• University of Delaware
• Supervised by
• Ping Xu and Todd Kitten
• Virginia Commonwealth University
• Tuesday, August 7, 2007

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Recap

• The rise of genomics
• What is reverse vaccinology?
• How does this help?
• Pan-genome

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Ultimate Goal

• Identify all possible vaccine candidates from
• Streptococcus mutans
• Compare with other
• candidates from other species
• of Streptococcus (ex.
• S. pyogenes,
• S. sanguinis) in the
• hopes of finding
• a universal vaccine

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The experiments performed in the past summer

• Isolate S. mutans genomic DNA
• Perform PCR amplification
• Perform DNA Sequencing
• Construct recombinant S. mutans mutant
• Transform DNA into S. mutans and E. coli
• Induce protein expression in E. coli
• Prepare surface protein by digestion for
  proteomics

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Recombinant PCR
Transposon protein
F1
R1
Kan cassette
A
B
Transposon protein
F2
R2
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Ladder Product
4000 base pair
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Sequencing and more
ATGGTTATAACGAAAGTACTTAAGTTAAAGCCAGCACATTTTTTTCTGGA
AGAGAAAAGTCTATATAGTTATCGGTATATGGTAA CTTATGGTGCCATT
CGAATATTGTATTCGGATTCTAANAAGAAGGCAGAATCTGGAACATTAAT
TGATTTGACAGGCGGTGGAT CTTGACGTTGTTTGATATCAAAGCNCGTG
TTTTTGATNATCAAATCATTATGAAA
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Transformation

• Homologous Recombination
• Kanamycin

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Colony PCR
Ladder C1 C2 C3 C4 C5
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Last step of this summer

• Surface digestion
• Peptide cleaving
• Proteomic analysis

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So, where will this go??
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Acknowledgments

• Dr. Ping Xu
• Dr. Todd Kitten
• Dr. Xiuchun Ge
• Joe Moeller
• The Kitten Lab
• Dr. Bucks Lab
• Jeff Elhai, BBSI participants, faculty and staff