INDUCIBLE TOXIN GENE CASSETTE FOR

Work presentationNishanti Sudhakar

Staphylococcus aureus

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

• 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

• 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

TOXIN-ANTI TOXIN SYSTEMS

LAC - OPERON

pKOR1

E. coli, S. aureus shuttle vector

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

pKOR1

AttB1 AttB2+

Rec. pKOR1

By product

Insertion of fragment into plasmid by Gateway technology

using BP clones

MAKING THE REC. PLASMID

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

Rec. pKOR1

S. aureus genome

Homologous recombination - Double crossover

By product+

IN-VIVO HOMOLOGOUS

RECOMBINATION

REPLACEMENT KNOCKOUT CASSETTE

pUC57

Knockout genome of cowan I

Double crossover

pSpaC promoter

PCN promoter

VACCINE STRATEGY

S. aureus carrying modified genome with

our MazF cassette

inhibitorlactose from milk in udder

= Immune response elicited+

Death of bacteriaToxin produced

SUBJECT EFFECTIVELY IMMUNE TO S. aureus

THANK YOU