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Team:TU-Delft/Sensing
From 2013.igem.org
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| + | <h4>Procedure</h4> | ||
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| + | <li> Grow 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. </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> Track the OD till 0.5 and then induce with AIP 1 µM and 10 µM. </li> | ||
| + | <li> After 3 hours of incubation time, check for GFP signals on the FACS (Fluorescence-activated cell sorting). </li> | ||
| + | </ol> | ||
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Revision as of 17:22, 3 October 2013
Sensing Device
Figure 1: Schematic diagram of the sensing part
In bacteria quorum sensing is an important mechanism for monitoring the state of the population. S. aureus uses its quorum sensing system to regulate a large part of its virulence genes, as having a large population is beneficial for S. aureus during infection. In this project we highjack the native S. aureus system and implement that in the antimicrobial peptide producing host E.coli.
We used the quorum sensing system of S. aureus in our peptidor in order to make sure the antimicrobial peptides will only be produced when S. aureus is present. This way there is less chance of other microbes becoming resistant against the peptides and therefore indirectly, through horizontal gene transfer, preventing S. aureus becoming resistant.
The system is composed out of the transmembrane receptor protein AgrC and the cytoplasmic protein AgrA. AgrA is phosphorylated by AgrC upon induction with auto-inducing peptide (AIP), after which it will act as a transcription factor positively acting on the promoter P2. AIP is made from the precursor peptide AgrD, which is circularized and secreted by AgrB (Figure 1).
Procedure
- Grow the construct BBa_K1022100 (pBAD AIP Receiver GFP) overnight.
- 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.
- Track the OD till 0.5 and then induce with AIP 1 µM and 10 µM.
- After 3 hours of incubation time, check for GFP signals on the FACS (Fluorescence-activated cell sorting).
