Week 39 (23rd-29th of September)

24th of September 2019

1. Third Estradiol bioactivity assay

Team members: Noel and Hitesh

After the unfortunate results before, we decided to increase the total Volume of each well to 200 µl, instead of the 140 µl we had used before. Also, we used tape to close the gap between lit and 96-well plate. Third, we reduced the incubation time to one night again.

Materials & Procedure

Different estradiol concentrations were tested, namely: 0, 4, 6, 8, 10, 12, 14, 16, 50, 100, 250, 500 pmol/liter.
In each well a total volume of 200 µl was pipetted, 175 µ of which was used for the cell culture (OD600 0.5). The remaining 25 µl was split between the necessary volume of the respective estrogen solution and water.
Both, “GPER-expressing” cells and control cells (transformed with an empty vector) were exposed to all the listed concentrations of estrogen. Each culture-concentration-combination was measured in tripletts.
The plate was left in the incubator for one night (30° C, 150 rpm). After this time, the fluorescence was measured.

Results

The results didn’t show a significant difference in fluorescence between Control and the GPER-expressing strain. We finally conclude that our GPER-Galphas-STE12-ZsGreen-strain is not able to produce ZsGreen in the presence of Estrogen in this type of experiment. Our best guess would be that the receptor is not expressed in the right membrane (which would be the plasma membrane) or in the wrong orientation. This means that estrogen, even though it can penetrate the membranes and bind to the receptor, doesn’t result in a transduction of the signal because the other parts other MAP Kinase is physically separated from the receptor.

2. Fluorescence Microscopy

Team members: Noel & Swenja

The following strains were analysed under the fluorescence microscope under the guidance of Professor Alexander Schulz:


Sample	Date of transformation	Colony number on the respective plate	Showed fluorescence before	Showed fluorescence this time
GPER-sfGFP	07/08	4	Yes	Yes
GPER-sfGFP	07/08	7	Yes	Yes
XLHCGR-sfGFP	07/08	3	No	Not tested
XLHCGR-sfGFP	29/08	1	Not tested	Yes
XLHCGR-sfGFP	29/08	2	Not tested	Yes
XLHCGR-sfGFP	29/08	3	Not tested	Not tested

Results

We could confirm the positive results of the GPER-sfGFP expressing colonies. Fortunately, the tested newer xLHCGR-sfGFP expressing colonies also showed the wanted green color.