work presentation oct 2015
DESCRIPTION
Work Presentation - NishanthiTRANSCRIPT
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INDUCIBLE TOXIN GENE CASSETTE FOR
Work presentationNishanti Sudhakar
Staphylococcus aureus
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CONTENTS1. Staphylococcus aureus
2. S. aureus in cattle
3. Toxin - antitoxin systems
4. Lac Operon
5. pKOR1
6. Construction of gene cassette
7. Knockout cassette
8. Vaccine strategy
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• Gram positive coccal bacterium. • Immobile, forms grape like clusters as
they divide in two planes. • Facultative anaerobes, non-sporulating,
catalase positive, coagulase positive. • Colonies are yellow. • Reproduces asexually by binary fission. • Has about 33% G+C content and an
average gene length of 824 nucleotides with 85% coding sequence.
• Responsible for various diseases including: mild skin infections, invasive diseases, and toxin mediated diseases.
• S. aureus also causes economically important mastitis in cows, sheep and goats.
Staphylococcus aureus
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• Leading cause of contagious mastitis in dairy cattle.
• Severe damage to milk producing tissue and leads to abscess formation.
• Generally results in a herd mastitis problem.
• The bacteria persist in mammary glands, teat canals, and teat lesions of infected cows. Mildly contagious. Spread from cow to cow primarily at milking time.
Staphylococcus aureus IN CATTLE
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TOXIN-ANTI TOXIN SYSTEMS
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LAC - OPERON
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pKOR1
E. coli, S. aureus shuttle vector
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CONSTRUCTION OF GENE CASSETTE
In S. aureus genomeToxin Anti-toxin
MazF Up flank MazF Dn flank
AttB1
AttB2
BamHI
Extension by PCR PCR product
AttB1AttB2
BamH
I
LigationAttB1 AttB2
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pKOR1
AttB1 AttB2+
Rec. pKOR1
By product
Insertion of fragment into plasmid by Gateway technology
using BP clones
MAKING THE REC. PLASMID
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Rec. pKOR1
DH10B
DC10B Cowan I
Transformation by CaCl2
Transformation by CaCl2
Transformed by electroporation
Rec. pKOR1
E. coli
E. coli S. aureus
Voltage: 21KV/cm Capacitance 25uf Resistance 100 ohms
Voltage: 21KV/cm TC: 1.1 msec
TRANSFORMATION
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Rec. pKOR1
S. aureus genome
Homologous recombination - Double crossover
By product+
IN-VIVO HOMOLOGOUS
RECOMBINATION
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REPLACEMENT KNOCKOUT CASSETTE
pUC57
Knockout genome of cowan I
Double crossover
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pSpaC promoter
PCN promoter
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VACCINE STRATEGY
S. aureus carrying modified genome with
our MazF cassette
inhibitorlactose from milk in udder
= Immune response elicited+
Death of bacteriaToxin produced
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SUBJECT EFFECTIVELY IMMUNE TO S. aureus
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THANK YOU