Team:TU-Delft/Protocol 10

Our project deals with E.coli cells which sense Auto-inducing peptides (AIPs) from the Staphylococcus aureus and starts producing Antimicrobial peptides in order to kill the Staphylococcus aureus. Different protocols used during the project are described below.

• Transforming Parts from Distribution kit
• Growing the Single Colonies from the Agar Plates
• Making glycerol stocks
• Plasmid Purification Protocol
• Restriction digestion
• Ligation
• Gel Extraction Procedure
• PCR Purification
• Tricine Gels
• General Peptide Production
• SUMO cleavage
• Lysis Protocol
• AIP Sensing Protocol
• Gene Design
• Primer Design

General Peptide Production

Procedure

1. Inoculate cells from a plate into 5mL of media. Grow overnight (o/n) at 37°C with rotation at 180 rpm.
2. In morning, dilute the o/n cultures 1/50 and follow the OD600 till it reaches 0.5-0.6.
3. Induce the cultures with 1mM final concentration of IPTG.
4. After 3 hrs of IPTG induction, 0.1 % of arabinose was added .
5. After 3 hrs of induction spin down the cultures at 6000 rpm for 10 min, RT.
6. Re-suspend the pellet in required amount of french press lysis buffer (give the composition)
7. Disrupt the cells using french press at 24000-26000 psi
8. Centrifuge the lysates at 45,000 rpm for 30 min to get rid of the cell debris. Collect the clear lysates and the pellet separately. Snap freeze them with liquid nitrogen and store the samples at -800C for further use.
9. Run the tricine gels (protocol)