Team:TU-Delft/Protocol 13

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<h4>Procedure: </h4>
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<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>
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<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) overnight at 37 degrees. </li>
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<li> Make 1/50 dilutions of the overnight grown culture. Check for OD at 600nm till 0.1.</li>  
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<li> Make 1/50 dilutions in LB medium of the overnight grown culture. Check for OD at 600nm till 0.1.</li>  
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<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>
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<li> 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.</li>
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<li> Check the OD till 0.5 and then induce with AIP to a final concentration of 1 µM and 10 µM. </li>
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<li> Check the OD at 600nm till 0.5 and then induce with AIP to a final concentration of 10 µM. </li>
<li> After 3 hours of incubation time, check for GFP signals on the FACS (Fluorescence-activated cell sorting). </li>
<li> After 3 hours of incubation time, check for GFP signals on the FACS (Fluorescence-activated cell sorting). </li>
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Latest revision as of 12:25, 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.


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).
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