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Team:TU-Delft/Protocol 13
From 2013.igem.org
Protocols
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:
- Grow the cells containing the construct BBa_K1022100 (pBAD AIP Receiver GFP) overnight at 37 degrees.
- Make 1/50 dilutions in LB medium of the overnight grown culture. Check for OD at 600nm till 0.1.
- 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.
- Check the OD at 600nm till 0.5 and then induce with AIP to a final concentration of 10 µM.
- After 3 hours of incubation time, check for GFP signals on the FACS (Fluorescence-activated cell sorting).
