Achievements
Medium
Prior to using our own device for detection of fluorescence in the sensor chips we used equipment readily available in the lab. A Molecular Imager® Gel DocTM XR+ from BIO-RAD was available which used UV light and incorporated two filters for the excitation ligth wavelength. Because of its limitted wavelengths of ligth available for excitation of fluorescent molecules the Gel DocTM XR+ was not ideal for our project. For exapample, it was possible to detect the expression of iLOV in our sensor chips, but in contrast detection of GFP was not possible.
(iLOV_GFP_HM_1,5h.png)
Concerning the medium used for our sensor chips, LB medium showed a high fluorescence background signal when exposed to UV light. The background fluorescence was to high to detect a signal emitted by our sensor cells thus we used minimal media (NA, M9, Hartman) in order to minimize the 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 on the minmal media.
(Chip_medium_geldoc.png)
There was the idea to store the chips at -20 °C, ready for use. But the cells died. Adding 5-10% glycerol resulted in an expression stop of fluorescence proteins. So the idea was rejected.
In our own device WatsOn with the optimized light (450/ 480 nm) the LB showed no fluorescence. The cells grow better there and so we use this now.
For longer storage up to 5 days in the fridge (+ 4°C) TB medium can be used. It has a smal background fluorescence in WatsOn.
(5Tage_K131026_neb_tb_1,5h)
Agar concentration
For the sensor chip an agarose concentration of 1.5% is used.
We first tried agar. When the agar concentration is lower than 1.5% the chip is not stable and brakes fast. When the agar concentration is higher than 1.5% the medium gets solid before pouring it into chip form.
In the end we choose agarose instead of agar because the agarose is better linked and so the diffusion is not so high.
Chip form
First we wanted to produce every chip individually. For a plane surface we wanted to use microscope slides. But the agar was to liquid and leaked. After that we made a closed form in which you can give the ager with a pipette. But this chips had big bubbles and were really difficult to produce.
(2_chipform.jpg)
In the end we use a self made open form. Here you produce 9 chips at the same time. You just have to cut them out (anhand) the lasered lines. This is a fast, easy and efficient way to produce chips. And because of the surface tension the chips are plane.
(final_chipform.jpg)
Induction
For the induction of the used constructs we use IPTG or 3-oxo-C12 HSL.
The sensor cells with K1319042 in BL21 can detect a IPTG concentration of 1 mM (0,2 µl).
(2µl_IPTG_1mM_K1319042_1h.png)
The sensor with the REACh constructs in BL21 can detect a IPTG concentration of ???
(Plate reader???)
The sensor cells with K131026 in BL21 can detect an HSL concentration of 500 µg/ml (0,2 µl).
It also can detect Pseudomonas aeruginosa.
(Zeitaufnahmen bearbeitet von Arne)
Testing our sensor Chips with a Platereader
Detecting the 3-oxo-C12 HSL with K131026 in our sensor chip with WatsOn.
Detecting Pseudomonas aeruginosa with K131026 in our sensor chip with WatsOn.
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