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Team:TU-Delft/Protocol 13
From 2013.igem.org
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<h4>Procedure: </h4> | <h4>Procedure: </h4> | ||
<ol> | <ol> | ||
| - | <li> Grow the cells containing the construct <a href="http://parts.igem.org/Part:BBa_K1022100" style="text-decoration: none"" target="_blank">BBa_K1022100</a> (pBAD AIP Receiver GFP, fig. 2) overnight. </li> | + | <li> Grow the cells containing the construct <a href="http://parts.igem.org/Part:BBa_K1022100" style="text-decoration: none"" target="_blank">BBa_K1022100</a> (pBAD AIP Receiver GFP, fig. 2) overnight at 37 degrees. </li> |
<li> Make 1/50 dilutions of the overnight grown culture. Check for OD at 600nm till 0.1.</li> | <li> Make 1/50 dilutions of the overnight grown culture. Check for OD at 600nm till 0.1.</li> | ||
<li> Then induce with 0.1% Arabinose. Keep aside samples with No Arabinose, No AIPs induction to use as control. BL21 cells and Const GFP are also used as negative and positive controls.</li> | <li> Then induce with 0.1% Arabinose. Keep aside samples with No Arabinose, No AIPs induction to use as control. BL21 cells and Const GFP are also used as negative and positive controls.</li> | ||
Revision as of 11:41, 4 October 2013
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, fig. 2) overnight at 37 degrees.
- Make 1/50 dilutions 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 cells and Const GFP are also used as negative and positive controls.
- Check the OD till 0.5 and then induce with AIP to a final concentration of 1 µM and 10 µM.
- After 3 hours of incubation time, check for GFP signals on the FACS (Fluorescence-activated cell sorting).
