Week 38 (16th-22nd of September)

19th September 2019

1. Second Estradiol bioactivity assay

Team members: Ojas, Iben and Noël

Preparation

The calculation of the first bioactivity were analysed. Thereby, it was found that the physiological saliva concentrations (between 4-16 pmol/l) were not met.

Therefore, new dilutions were prepared:

First 27.2 mg estradiol were diluted in 1 ml of ethanol resulting in a 0.1 mol/l solution. For the experiments a 10 to the power of 3, 6, 7, 8 and 9 were prepared.

Materials

The aforementioned estrogen dilutions

The S4C3 Yeast GPER-Galphas-STE12-ZsGreen culture from the 12th September

Control culture that had an empty plasmid integrated into its genome

96 well plate

Glu-U media

Procedure

Similar to first attempt that was conducted on the 16th 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 140 µl was pipetted, 120 of which was used for the cell culture (OD600 0.5). The remaining 20 µl was split between the necessary volume of the respective estrogen solution and water

Both, “GPER-expressing” cells and control cells 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 three nights (30° C, 150 rpm)

Results

Because of a systematic error, the fluorescence in each well was to high and it was impossible to analyse the data properly.>/p>

20th September 2019

Team members: Ojas, Iben and Noel

The calculation of the first bioactivity were analysed. Thereby, it was found that the physiological saliva concentrations (between 4-16 pmol/l) were not met. Therefore, new dilutions were prepared: First 27.2 mg estradiol were diluted in 1 ml of ethanol resulting in a 0.1 mol/l solution. For the experiments a 10 to the power of 3, 6, 7, 8 and 9 were prepared.

Materials

The aforementioned estrogen dilutions

The S4C3 Yeast GPER-Gαs-STE12-ZsGreen culture from the 12th September

A control culture that had an empty plasmid integrated into its genome

A 96 well plate

Glu-U media

Procedure

Similar to first attempt that was conducted on the 16th 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 140 µl was pipetted, 120 of which was used for the cell culture (OD600 0.5). The remaining 20 µl was split between the necessary volume of the respective estrogen solution and water. Both, “GPER-expressing” cells and control cells 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 three nights (30° C, 150 rpm). After this time had past, the ZsGreen amount was assessed via the fluorescence of this protein.

Results

Unfortunately, the results were unusable because the three nights were a too long incubation time and the ELISA plates had unfortunately dried out. The following solutions for this problem were discussed:

Reduce the incubation time to one night
Seal the gap between lit and plate with tape that cannot be penetrated by gaseous water
Have a greater volume in each well (and bigger wells) so a little volume change doesn’t have a big influence.

With this in mind, the assay was repeated a third time.