Interlab is a peculiar work that aims to decrease lab to lab variety. Due to different conditions in labs, the results obtained from years of work are sometimes considered unreliable. Thus, it is important to improve the measurement tools available for everyone. In order to support this project, we followed the standard iGEM protocol. This protocol is used to calculate the difference between OD and CFU. GFP and fluorescein was used in the experiments and measurements with suggested tools.
We began Interlab Measurements in 25.06.2018 Ph. D. student Evrim Elçin helped us with the protocols and constructing our experiments.
In order to convert our density readings to OD600, we constructed the first experiment of the Plate Reader and CFU Protocol. The given LUDOX CL-X (45% colloidal silica suspension) were added into wells A1, B1, C1, D1 in the amount of 100 μL. Then, we added 100 μL of dd H2O into wells A2, B2, C2, D2 and measured the absorbance at 600 nm of all samples in Thermo Scientific Multiskan Go spectrophotometer.
After the first experiment of the protocol, we constructed an experiment to find Particle Standard Curve with the use of Microspheres. Thus, we prepared a dilution series of monodisperse silica microspheres by following the Microsphere Protocol. We used 300 μL of Silica beads, dd H2O and a white 96 well plate with clear flat bottom and measured the absorbance at 600nm. All the settings of the spectrophotometer and materials used were same with the ones in the first experiment.
Fluorescein Standard Curve
The third measurement of the Protocol was fluorescein standard curve. Because the fluorescein that was supplied by iGEM was sensitive to light, we used a black 96 well plate with clear flat bottom. We have payed extreme attention to the sensitivity of the fluorescein while constructing the experiment. Thus, we conducted it with minimum light exposure as possible. At first, we prepared a fluorescein stock solution with 1x PBS and 10x fluorescein. Then, we began the serial dilutions of fluorescein in 96 well plate to measure fluorescein in the plate reader with the settings suggested by iGEM.
CELL Measurement Protocol
After we were finished with all the calibration protocols, we moved on to the Cell Measurement protocol. The calibration protocols made us feel certain about the measurement of both fluorescence and OD600. Hence, we immediately began to perform our experiments as suggested in iGEM Interlab Plate Reader Protocol. On our first day, we transformed the given plasmids into our competent E. coli DH5-α with the help of iGEM Transformation protocol. Therefore, our bacteria were grown perfectly so we were able to pick two colonies from each of the transformation plates. We especially payed attention to select the colonies which were away from the others. Then, we inoculated our single colonies in LB medium and Chloramphenicol. Our cells were grown overnight (16-18h) at 37 degrees Celsius and 220 rounds per minute in LB medium and Chloramphenicol. The blur in the broth was the assurance that the plasmids were successfully transformed.
The last day was a little confusing and hard but we were able to construct the experiment according to the given protocol. At first, we made a 1:10 dilution of our overnight cultures in LB+Chloramphenicol. Then measured the OD600 values of the diluted cultures. Thanks to the results, we were able to set the OD600 values to 0.02 in a final volume of 12 ml LB medium + Chloramphenicol by diluting further in 50 ml falcon tube. In order to be sure that our last diluted colonies had an OD600 of 0.02, we measured them and observed that the dilution worked properly. The next step was taking 500 μL samples of the each diluted cultures at 0 hours and 6 hours. Thus we measured OD600 of 32 samples and imported the data into Excel sheet. Our plate reader’s setting was 25 degrees Celsius at 600nm and the pathway correction was turned off.
Furthermore, we were so ambitious to finish the protocol and make it perfect. Thus, we specially tried to prevent every single contamination risk. We worked in a hood and close to fire. Every material we used such as pipette tips and Eppendorfs were sterilized.
After we were sure that we were working on a sterile environment and we were ready, the starting sample preparation of Colony Forming Units per 0.1 OD600 E.coli cultures protocol was set. We made a 1:10 dilution of the cell cultures of positive controls and negative controls. Then we measured the OD600 of the cultures and used LB + Chloramphenicol as blank media. The next step was just calculations. The cultures were diluted to 0.1 ABS at 600 nm in 1 ml LB + Chloramphenicol for each culture of each Control device. At the end, we had three starting samples of each control devices’ cultures.
The math was perfect but it was just the beginning. We made a serial dilution of all the starting samples. It took us so long. We prepared three tubes of 1900 μL LB + Chloramphenicol and two tubes of 900 μL LB + Chloramphenicol for each serial dilutions. Then started from the beginning and add 100 μL starting sample into 1900 μL LB + Chloramphenicol. The serial dilution was constructed as suggested in the protocol. These steps were done for each starting samples. After the serial dilutions, we spread plate 100 μL on LB + Chloramphenicol for Dilutions 3,4,5 and incubate at 37 degrees Celsius overnight. After approximately 20 hours of incubation, we counted the colonies and multiplied by the final dilution factor. The results were imported into plate reader forms, and submitted to iGEM.Click to see cell measurement results