Team:Aachen/Project/2D Biosensor
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{{Team:Aachen/FigureFloat|Aachen_ILOV_GFP_HM_1,5h.png|title=iLOV and GFP in the Gel Doc<sup>TM</sup>|subtitle=Sensor cells producing iLOV (A) and GFP (B) 1.5 h after induction.|left|width=500px}} | {{Team:Aachen/FigureFloat|Aachen_ILOV_GFP_HM_1,5h.png|title=iLOV and GFP in the Gel Doc<sup>TM</sup>|subtitle=Sensor cells producing iLOV (A) and GFP (B) 1.5 h after induction.|left|width=500px}} | ||
- | Our first approach (before developing our own device) was to use the Molecular Imager® Gel Doc™ XR+ from BIO-RAD in our lab to detect fluorescence, | + | Our first approach (before developing our own device) was to use the Molecular Imager® Gel Doc™ XR+ from BIO-RAD in our lab to detect fluorescence, which uses UV and white light illuminators. However, only two different filters were available for the excitation light wavelength, which resulted in very limited possibilities for the excitation of fluorescent molecules. For example, it was possible to detect the expression of iLOV in our sensor chips, but in contrast the detection of GFP was not possible. Hence, the '''Gel Doc™ was not suitable for our project'''. |
{{Team:Aachen/FigureFloat|Aachen_Chip_medium_geldoc.png|title=Differend medium in the Gel Doc™|subtitle=complex media exhibited high background fluorescence while less back-ground fluorescence was observed with the minimal media (HM, M9, NA).|right|width=500px}} | {{Team:Aachen/FigureFloat|Aachen_Chip_medium_geldoc.png|title=Differend medium in the Gel Doc™|subtitle=complex media exhibited high background fluorescence while less back-ground fluorescence was observed with the minimal media (HM, M9, NA).|right|width=500px}} | ||
{{Team:Aachen/FigureFloat|Aachen_5days_K131026_neb_tb_1,5h.jpg |title=Testing our chips' shelf-life|subtitle= [http://parts.igem.org/Part:BBa_K131026 K131026] in NEB induced after 5 days of storage at 4°C. The right chip was induced with 0.2 µL of 500 µg/mL HSL, and the image was taken after 1.5 h.|left|width=500px}} | {{Team:Aachen/FigureFloat|Aachen_5days_K131026_neb_tb_1,5h.jpg |title=Testing our chips' shelf-life|subtitle= [http://parts.igem.org/Part:BBa_K131026 K131026] in NEB induced after 5 days of storage at 4°C. The right chip was induced with 0.2 µL of 500 µg/mL HSL, and the image was taken after 1.5 h.|left|width=500px}} | ||
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<!--Regarding the medium used for our sensor chips, LB medium showed a high background fluorescence when exposed to UV light in the Gel Doc. Surprisingly, the background fluorescence resulting from the LB medium was too high to detect a signal emitted by our sensor cells. Hence, minimal media (NA, M9, Hartman (HM)) was used to minimize background fluorescence, but this approach resulted in less to no growth of our sensor cells. In our device ''WatsOn'', optimized wavelengths of 450 nm and 480 nm were used for excitation of iLOV and GFP, respectively. When exposed to either excitation wavelength TB medium, which is basically an improved LB medium and highly supports cell growth, showed strong background fluorescence in our own device. High background fluorescence was also observed for TB medum when using the Gel Doc. In contrast to the Gel Doc LB medium showed minimal fluorescence in our device ''WatsOn'' and no difficulties in cultivation of our ''Cellocks'' were observed. Because of the reduced fluorescence compared to TB medium when using ''Watson'' for sensor chip evaluation and because of sufficient cultivation conditions for our 'Cellocks'' LB medium was chosen over TB mediium for sensor chip manufacturing. --> | <!--Regarding the medium used for our sensor chips, LB medium showed a high background fluorescence when exposed to UV light in the Gel Doc. Surprisingly, the background fluorescence resulting from the LB medium was too high to detect a signal emitted by our sensor cells. Hence, minimal media (NA, M9, Hartman (HM)) was used to minimize background fluorescence, but this approach resulted in less to no growth of our sensor cells. In our device ''WatsOn'', optimized wavelengths of 450 nm and 480 nm were used for excitation of iLOV and GFP, respectively. When exposed to either excitation wavelength TB medium, which is basically an improved LB medium and highly supports cell growth, showed strong background fluorescence in our own device. High background fluorescence was also observed for TB medum when using the Gel Doc. In contrast to the Gel Doc LB medium showed minimal fluorescence in our device ''WatsOn'' and no difficulties in cultivation of our ''Cellocks'' were observed. Because of the reduced fluorescence compared to TB medium when using ''Watson'' for sensor chip evaluation and because of sufficient cultivation conditions for our 'Cellocks'' LB medium was chosen over TB mediium for sensor chip manufacturing. --> | ||
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=== Optimal agarose concentration for sensor chip manufacturing === | === Optimal agarose concentration for sensor chip manufacturing === | ||
For the sensor chip manufacturing, agarose was preferred over agar, because of a uniform linkage that results in a better chip homogeneity. In addition, agarose reduced diffusion of the inducer molecules through the chip. A reduced diffusion is vital to observe distinct fluorescent spots on the sensor chips. | For the sensor chip manufacturing, agarose was preferred over agar, because of a uniform linkage that results in a better chip homogeneity. In addition, agarose reduced diffusion of the inducer molecules through the chip. A reduced diffusion is vital to observe distinct fluorescent spots on the sensor chips. |
Revision as of 18:17, 17 October 2014
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