Team:TU-Delft/Protocol 13

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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

AIP Sensing Protocol

Procedure:

1. Grow the cells containing the construct BBa_K1022100 (pBAD AIP Receiver GFP) overnight at 37 degrees.
2. Make 1/50 dilutions in LB medium of the overnight grown culture. Check for OD at 600nm till 0.1.
3. Then induce with 0.1% Arabinose. Keep aside samples with No Arabinose, No AIPs induction to use as control. BL21(DE3) cells and cells constitutively expressing GFP are also used as negative and positive controls.
4. Check the OD at 600nm till 0.5 and then induce with AIP to a final concentration of 10 µM.
5. After 3 hours of incubation time, check for GFP signals on the FACS (Fluorescence-activated cell sorting).