Team:TU-Delft/Protocol 1

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

Transforming Parts from Distribution kit

Requirements:

1. Distilled water
2. Pipettes
3. Ice
4. Competent cells
5. SOC media
6. Agar plates with Antibiotics
7. Timer
8. Water bath

Prodecure

1. Mark the location on distribution plate by looking at the right row and column as mentioned. Punch the plate with a pipette and add 10 µL of distilled water.
2. Resuspend the Dry DNA on the distribution plate with distilled water.
3. Inoculate the competent cells with the desired DNA from the distribution plate as described in step 2. Add 1 µL of the resuspended DNA to the competent cells. Use 2 mL tubes and keep the cells on ice at all the times for a more efficient transformation.
4. Incubate the transformation on ice for 5 min.
5. Heat shock the transformation the water bath at 42⁰C for 30 sec.
6. Incubate on ice for 2 min.
7. Add 200 µL SOC Media to the transformation.
8. Incubate the transformation at 37⁰C for 1 hour.
9. Plate out 20 µL of transformation with 10% and 90% on separate agar plates with appropriate antibiotics.
10. Incubate the agar plate overnight (14-16 hours) at 37⁰C.

Single colonies are selected from the above plates and grown in LB media to prepare glycerol stocks and carry out Miniprep to extract the desired plasmid.