Background
The InterLab Study of this year aims to analyze the connection between the measurement of the Optical Density of E. coli and the GFP in the culture. This proportionality has differed in labs around the world in the past years and we are now trying to approach the results by using the protocol provided by iGEM. This year's major question was: “Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-farming units (CFUs) instead of OD?“
Calibrations
At first, we measured the OD600 Reference point according to the LUDOX-Protocol. This included registering the absorbance of LUDOX CL.X and ddH2O at 600 nm.
Partide Standard Curve
For the Partide standard curve, we followed the Microsphere-Protocol, for which we started by mixing 96 µl of microspheres with 904 µl of ddH2O forming the microsphere stock solution. In the next step, we performed a serial dilution of this stock solution with 4 technical replicates through 11 wells each. After this, we measured the Abs600 of all samples, collected in the following table.
Fluorescence Standard Curve
Next, to form the Fluorescence Standard Curve, we executed the Fluorescein-Protocol, according to which we started by resuspending the provided fluorescein in 1 ml of 1xPBS, resulting in a 10xfluorecein stock solution. To lower the concentration of fluorescein we added 100 µl of the 10xfluorescein stock solution afterward to 900 µl of 1xPBS, making it a 1xfluorescein stock solution. Following this, we again performed a serial dilution of 4 technical replicates through 11 wells, after which we measured the fluorescence of all samples.
Cell Measurement Protocol
For completing the Cell Measurement-Protocol we began by transforming 6 different devices, containing plasmids which are all found in pSB1C3, of E. coli DH5 as well as a positive and a negative control on a plate. After letting the plates grow overnight we picked two colonies from each plate which we inoculated in 5-10 ml of LB medium and Cloramphenicol. We inoculated for 16-18 hours at 37 °C and 220 rpm. The next day we added 0.5 ml of each culture to 4.5 ml of LB and Chlor. we diluted to a 1:10 dilution every of which we measured the Abs600 values. We then put 500 µl of every sample at hour 0 on the ice and then inoculated the remainder of the cultures for another 6 hours. At this point, we also put 500µl of each culture in hour 6 on ice. Finally, we measured the fluorescence as well as the Abs600 of both colonies of all 6 devices and the positive and negative control at hour 0 and hour 6.
Colony Forming Units per 0.1 OD600 E.coli cultures
To prepare the Starting Sample we formed overnight cultures of two positive and two negative control cultures diluted 1:8 in LB+Cam. Each culture included 200 µl and was pipetted into a 96-Well plate as well as 2 200µl wells with Blank Media. In the next step, the cultures were diluted to OD600=0.1 in 1 ml of LB+Cam with 3 technical replicates of each well filled with positive and negative control cultures. These formed the Starting Samples.
Next, we performed the dilution series: first we diluted the Starting Samples 1:20 into Dilution 1. We then diluted this dilution 1:20 into Dilution 2. The third dilution was formed by diluting 1xDilution 2 into 20x LB+Cam. This dilution with the final dilution factor 8x10 4 was spread onto an LB+Cam plate. After this, we diluted Dilution 3 1:10 into Dilution 4 which was afterwards also spread on a plate. This culture had the final dilution factor 8x10 5. Lastly, we diluted Dilution 4 once more 1:10. We also spread the hereby formed Dilution 5 (final dilution factor 8x10 6)on a plate. All 36 (12x3) plates were then incubated at 37°C for 20 hours.
After these steps, we counted all colonies on every plate and multiplied these counts by the final dilution factor of each plate.
