Team:Aachen/Project/2D Biosensor
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=== Medium === | === Medium === | ||
Prior to using our own device for detection of fluorescence emitted by the sensor chips we used equipment readily available in the lab. A Molecular Imager® Gel Doc<sup>TM</sup> XR+ from BIO-RAD was available which uses UV and white light illuminators. However, only two different filters were available for the excitation ligth wavelength, which resulted in very limitted possibilities for the excitation of fluorescent molecules. For example, it was possible to detect the expression of iLOV in our sensor chips but the detection of GFP was not possible. It was thus determined that the Gel Doc<sup>TM</sup> XR+ was not suitable for our project. | Prior to using our own device for detection of fluorescence emitted by the sensor chips we used equipment readily available in the lab. A Molecular Imager® Gel Doc<sup>TM</sup> XR+ from BIO-RAD was available which uses UV and white light illuminators. However, only two different filters were available for the excitation ligth wavelength, which resulted in very limitted possibilities for the excitation of fluorescent molecules. For example, it was possible to detect the expression of iLOV in our sensor chips but the detection of GFP was not possible. It was thus determined that the Gel Doc<sup>TM</sup> XR+ was not suitable for our project. | ||
- | + | {{Team:Aachen/Figure|Aachen_iLOV_GFP_HM_1,5h.png|title=iLOV and GFP in the Gel Doc<sup>TM</sup>|subtitle=Cells producing A) iLOV B) GFP |width=300px}} | |
Regarding the medium used for our sensor chips, LB medium showed a high background fluorescence when exposed to UV light. Surprinsingly, the background fluorescence resulting from the LB medium was to high to detect a signal emitted by our sensor cells. Instead we tried using minimal media (NA, M9, Hartman) in order to minimize background fluorescence. The appliction of minimal media was sufficient to minimize the background fluorescence, but this approach came with the drawback of minimal to zero growth of our sensor cells. | Regarding the medium used for our sensor chips, LB medium showed a high background fluorescence when exposed to UV light. Surprinsingly, the background fluorescence resulting from the LB medium was to high to detect a signal emitted by our sensor cells. Instead we tried using minimal media (NA, M9, Hartman) in order to minimize background fluorescence. The appliction of minimal media was sufficient to minimize the background fluorescence, but this approach came with the drawback of minimal to zero growth of our sensor cells. | ||
- | + | {{Team:Aachen/Figure|Aachen_Chip_medium_geldoc.png|title=Differend medium in the Gel Doc<sup>TM</sup>|subtitle= high background fluorescence of the full medium (LB) and no background fluorescence of the minimal medium|width=900px}} | |
- | Further experiments were conducted to test long-time storage of the sensor chips. Storage at -20 °C resulted in the loss of our sensor cells. Adding 5-10% (v/v) glycerol ensured survival of the sensor cells, but resulted in an expression stop of fluorescence proteins. Thus, the idea of long time storage of the sensor chips had to be passed on. However, it was possible to store ready-to-use sensor chips for 2 days at 4 °C when using LB medium and storage for 5 days was possible with chips made from TB medium. | + | Further experiments were conducted to test long-time storage of the sensor chips. Storage at -20 °C resulted in the loss of our sensor cells. Adding 5-10% (v/v) glycerol ensured survival of the sensor cells, but resulted in an expression stop of fluorescence proteins. Thus, the idea of long time storage of the sensor chips had to be passed on. However, it was possible to store ready-to-use sensor chips for 2 days at 4 °C when using LB medium and storage for 5 days was possible with chips made from TB medium. |
+ | {{Team:Aachen/Figure|Aachen_5Tage_K131026_neb_tb_1,5h.png|title=K131026 in NEB induced after 5 days |subtitle= after storage for 5 days at 4 °C the chips were induced with 0.2 µl of 500nbsp;µg/ml HSL, image taken after 1.5 h |width=300px}} | ||
- | 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 the | + | 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 the TB medium showed minimal background fluorescence and no difficulties were observed in cultivation of our ''Cellocks''. Furthermore, a reduction of background fluorescence compared to TB medium was observed when using LB medium for sensor chip manufacturing in combination with fluorescence evaluation using ''WatsOn''. |
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=== Agar Concentration === | === Agar Concentration === |
Revision as of 12:18, 16 October 2014
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