Team:Aachen/Project/2D Biosensor
From 2014.igem.org
m (→Principle of Operation) |
(→Development & Optimization) |
||
Line 116: | Line 116: | ||
=== 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> 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> 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= | + | |
+ | {{Team:Aachen/Figure|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.|width=900px}} | ||
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 less background fluorescence of the minimal medium|width=900px}} | {{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 less background fluorescence of the minimal medium|width=900px}} | ||
- | {{Team:Aachen/FigureFloat|Aachen_5days_K131026_neb_tb_1,5h.jpg |title=K131026 in NEB induced after 5 days | + | {{Team:Aachen/FigureFloat|Aachen_5days_K131026_neb_tb_1,5h.jpg |title=Testing our chips' shelf-life.|subtitle= 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 1.5 h afterwards.|width=300px}} |
+ | |||
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. | ||
Revision as of 16:16, 16 October 2014
|
|
|
|
|