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work presentation oct 2015 .pdf


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Title: work presentation oct 2015

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INDUCIBLE TOXIN GENE
CASSETTE FOR
Staphylococcus aureus
Work presentation
Nishanti Sudhakar

CONTENTS
1. 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

Staphylococcus aureus


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 IN CATTLE






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.

TOXIN-ANTI TOXIN SYSTEMS

LAC - OPERON

pKOR1

E. coli, S. aureus shuttle vector

CONSTRUCTION OF GENE CASSETTE
Toxin

Anti-toxin

In S. aureus genome
MazF Up flank

MazF Dn flank

Ba
Extension by PCR

AttB2

HI

AttB1

Bam

mH
I

AttB1

PCR product

Ligation
AttB1

AttB2

AttB2

MAKING THE
REC. PLASMID
pKOR1

Rec. pKOR1

AttB1

AttB2

Insertion of fragment into
plasmid by Gateway technology
using BP clones

+
By product


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