Team:Tartu TUIT/InterLab

Our team has decided to participate in the 5th International InterLaboratory Measurement Study in synthetic biology(Interlab). The goal of the Interlab is to standardise the measurement procedure for green fluorescent protein, so that it is possible for laboratories from all over the world to share and compare the obtained results.
We are excited to have an opportunity to contribute to this study.

This year, the question that participating teams are trying to answer is if it is possible to «reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?»

Throughout the course of this study, we have been following the protocols from the iGem Interlab Study page.

We have measured OD600 and GFP fluorescence of 6 different test devices along with positive and negative control. Cells were grown in the LB+Chloramphenicol media and diluted to the 0.01 OD. Measurements were conducted right after the dilution and after the 6 hours incubation at 37°C, 220 rpm.

Our results are presented on Figures 1,2,3,4:

Figure 1.A graph representing the uM Fluorescein to OD ratio before the incubation
Figure 2.A graph representing the uM Fluorescein to OD ratio after the 6 hours incubation
Figure 3.A graph representing the MEFL to particle ratio before the incubation
Figure 4.A graph representing the MEFL to particle ratio after the 6 hours incubation

Our team has encountered some problems. Test Devices 1 and 5 did not show the sufficient growth after the 6-hours of incubation despite overnight cultures growing well. After repeating the protocol several times we still did not observe the increase in OD600 during 6 hours growth. When the cultures were left overnight they have reached the growth saturation. This fact eliminated the possibility of pipetting mistake.

Although all the measurements should have been obtained at room temperature (20-25 C), we have done all the measurements at 30 C. That happened due to the hot weather and the absence of cooling device inside the microplate reader.

Some other complications we encountered were connected to the CFU protocol. The colony growth was low and, therefore, the measurements were inaccurate. We have redone the experiments several times. We have tried to troubleshoot this problem by changing the antibiotics concentration in the plates, raising OD of the starting sample, increasing the volume of the culture spread on the plates. However, this did not improve the results.

Interlab became a valuable experience for our team. We hope that our measurements along with the data from other teams will be useful for the study and look forward to seeing the results of this year.