Team:Aachen/Project/2D Biosensor
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=== Testing our Sensor Chips in a Platereader === | === Testing our Sensor Chips in a Platereader === | ||
- | {{Team:Aachen/FigureFloat|Aachen_K1319042_Platereader.gif|title=Testing K1319042 in our sensor chips|subtitle=K1319042 in our | + | {{Team:Aachen/FigureFloat|Aachen_K1319042_Platereader.gif|title=Testing K1319042 in our sensor chips|subtitle=K1319042 in our sensor chip induced with 2 µL IPTG and measured with a plate reader. Blue color indicates no fluorescence, red color indicates fluorescence. Top chip is not induced, bottom chip is induced with IPTG.|width=300px}} |
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To establish a proof of principle we used our construct [http://parts.igem.org/Part:BBa_K1319042 K1319042] an IPTG inducible iLOV. They were introduced into our sensor chips and then fluorescence was measured every 15 minutes after an induction with 2 µl 100 mM IPTG. | To establish a proof of principle we used our construct [http://parts.igem.org/Part:BBa_K1319042 K1319042] an IPTG inducible iLOV. They were introduced into our sensor chips and then fluorescence was measured every 15 minutes after an induction with 2 µl 100 mM IPTG. | ||
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=== Detecting 3-oxo-C{{sub|12}} HSL with sensor chips === | === Detecting 3-oxo-C{{sub|12}} HSL with sensor chips === | ||
- | {{Team:Aachen/FigureFloatRight|Aachen_K131026_Platereader.gif|title=Testing K131026 in our sensor chips|subtitle=K131026 in our | + | {{Team:Aachen/FigureFloatRight|Aachen_K131026_Platereader.gif|title=Testing K131026 in our sensor chips|subtitle=K131026 in our sensor chip induced with 0.2 µL 3-oxo-C{{sub|12}} HSL and measured with a plate reader. Blue color indicates no fluorescence, red color indicates fluorescence.|width=300px}} |
- | As a next step we used [http://parts.igem.org/Part:BBa_K131026 K131026] from the 2008 iGEM Team | + | As a next step, we used [http://parts.igem.org/Part:BBa_K131026 K131026] from the 2008 iGEM Team Calgary in our sensor chips to detect 3-oxo-C{{sub|12}} HSL which is produced by ''Pseudomonas aeruginosa'' during quorum sensing. First, we tested them by direct induction with purified 3-oxo-C{{sub|12}} HSL (0.2 µL, 500 µg/mL). A fluorescence measurement was taken every 15 min with an excitation wavelength of 496 nm and an emission wavelength of 516 nm (for GFP). |
- | The measured fluorescence showed | + | The measured fluorescence again showed a distinct signal on the induced chip (bottom) compared to the uninduced chip (top). The fluorescence clearly starts in the middle of the chip (point of induction) and then extends outwards, still showing an ever increasing signal of fluorescence. The base level of fluorescence, even though a lot lower than the bottom chip, is attributed to leakiness of the promoter and general background fluorescence of growing ''E. coli'' cells. The difference between the induced and non-induced chips indicates a clear response to the HSL and a proof for the ability of our sensor chip design to being able to detect the HSL produced by ''Pseudomonas aeruginosa''. |
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Revision as of 17:02, 16 October 2014
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