Team:Tuebingen/InterLab
InterLab
Just Why?- Manu
Introduction
As a kickoff for our work in the lab, our team decided to take part in this year’s international InterLab Measurement Study, intending to improve measurement tools and to develop a solid and repeatable measurement protocol, making the comparison of measurements carried out in different labs more straightforward. Since being one of the most utilized markers all over the world, the fluorescent protein GFP (Green Fluorescent Protein) was chosen as a measurement marker.
In order to contribute to the 2018 InterLab Measurement study, the participating teams were advised to measure the OD600 and the fluorescence of 8 different constructs expressed in DH5a E.colis on a plate reader. Additionally, a protocol to calibrate OD600 to colony forming units (CFU) counts, which directly refer to the cell concentration in the culture, was provided.
Materials and Methods
2.1 Materials
Plate Reader: Tecan Infinite M200 Pro
96 well plates (white): Source unknown
DH5a Escherichia Coli
glass test tubes
2.2 Devices
Hier muss eine Tabelle rein
2.3 Workflow Cell Measurements
Hier muss der Workflow rein
Measurements
3.1 Calibration Measurements
All calibration measurements were performed as suggested by the protocol.
3.1.1 OD600 Reference Point– Ludox CL-X
Hier muss die Ludox Tabelle rein
3.1.2 Particle Standard Curve – Microspheres
Bild Particle Standard Curve linear
Bild Particle Standard Curve log
3.1.3 Fluorescence Standard Curve – Fluorescein
Bild Fluorescence Standard Curve linear
Bild Flourescence Standard Curve log
3.2 Cell Measurements
3.2.1 Transformation of 8 Test Devices
The transformation was carried out according to the iGEM Protocol. The clones were picked and treated as suggested by the cell measurement workflow (Figure 1).
3.2.2 OD600 Measurement
The OD600 of all samples was measured at 0h and after 6h of incubation at 37°C. All samples started with similar ODs of about 0.03 to 0.04. After 6h an increase of the OD was noticeable in all strains. The negative control (LB + Amp) did not show a change of OD within the course of the experiment (Figure 4).
Bild OD600 results
3.2.3 Fluorescent Measurement
Furthermore, the fluorescence of the strains with the 8 different devices was measured after 0h and after 6h of incubation. The negative control showed, as expected, very low levels of fluorescence, whereas the positive control showed in comparison to that, much more fluorescence. However, contrary to our expectations, the positive control showed less fluorescence after 6h as at time point 0. The same is true for the devices 3, 4 and 5. However, the values of the devices 3 and 5 correspond to the negative control, from which it can be concluded that these devices do not show fluorescent activity. In our experiment the devices 1 and 2 show the highest fluorescent values after 6h of incubation.
Bild Fluorescent results
3.2.4 CFU Protocol
Hier ist ein Text. Lol Lol Lol.
Discussion
During the performance of the fluorescent measurement of the cells it became clear, that the utilized plate reader did not work properly. For this reason, we had to repeat the fluorescent measurements with a different plate reader on a different day. However, we tried to make sure, that all measurements still are as comparable as possible by using the same plate reader model and the same plates.
After sending in our data and informing iGEM of the difficulties we were encountering, we received the message, that even though our data does not match the iGEM measurement standards, we will still be awarded with the Broze medal standard for our many tries to handle the problems correctly. For this we want to thank the iGEM committee very much.