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InterLab

This is the first year that a team from Technion participated in the Interlab Study.
The goal of this experiment was to establish a GFP measurement protocol based on engineering principles using a plate reader.
We are hoping that the data we have provided will help in developing novel characterization methods in the rapidly growing fields of biological design and synthetic biology.

We tested 8 different plasmids (six devices, a positive control and a negative control). All parts were cloned into the pSB1C3 vectors that were provided in the distribution kit.
All steps below were conducted according to the plate reader protocol as was instructed to us.

Methodology and results

OD600 Reference point

The first step was to use LUDOX-S40 (will referred from now as LUDOX) as a single point reference to obtain a ratio metric conversion factor to convert the absorbance data into a standard OD600 measurement.

We measured 4 replicates of LUDOX and used H2O absorbance as a blank.
The corrected absorbance of LUDOX was calculated as 0.001107. Correction factor was then calculated by dividing the corrected absorbance by a reference OD value 0.0425. Hence, the correction factor is 3.837 (Figure 1).

Figure 1: Absorbance measurement of LUDOX 100% and H2O and correction factor table

Fluorescein fluorescence standard curve

In order to generate a standard curve of fluorescence depending on fluorescein concentration we prepared a dilution series of fluorescein in 4 replicates and measured the fluorescence in a 96 well plate in our plate reader (Tecan infinite 200 pro). We diluted the fluorescein stock with 1XPBS into 12 different concentration.
We used a filter with excitation wavelength 495 nm and emission wavelength 535 nm.

Figure 2: Fluorescein standard curve plotted from the data of 12 concentration of 4 replicates

Cell measurement

Day 1: we began by transforming (transformation protocol ) Escherichia coli DH5α with the following plasmids:

Positive control
Negative control
Test Device 1: J23101+I13504
Test Device 2: J23106+I13504
Test Device 3: J23117+I13504
Test Device 4: J23101.BCD2.E0040.B0015
Test Device 5: J23106.BCD2.E0040.B0015
Test Device 6: J23117.BCD2.E0040.B0015

Day 2: we picked two colonies from each plate and inoculated them into 5 ml LB + Chloramphenicol, and then grew the cells overnight at 37°C and 220 rpm.

Day 3: we measured an OD of 600 from the overnight cultures and diluted the cultures to a target OD of 600 of 0.02 in 12 ml LB + Chloramphenicol in 50 ml falcon tube covered with foil to block light (Figure 3).
After diluting the cultures we incubated them at 37°C and 220 rpm.

Figure 3 : The diluted cultures in the covered falcon tubes

We then took 500 μl samples of the cultures at 0, 2, 4, and 6 hours of incubation. At each time point we measured the OD and Fl of the samples, 4 replicates from each colony.
All the data was summarized and submitted to the measurement committee.

Figure 4 : Layout for Abs600 and Fluorescence measurement

To conclude, participating in the Interlab experiment was a fun and teaching experience. We are happy that we had the chance to contribute to this important measurement study and to the iGEM community.