The 3rd International InterLab Study of the iGEM competition sought to compare the results of certain measurements conducted by on different devices by different scientists in different labs all over the world. In order to have a distinctive set of measurable parameters, the expression of GFP (I13504) under the control of three promotors with different strengths was subject to measurement. The promotors are part of a constitutive promotor family and where constructed by John Anderson in 2006 and added to the iGEM Registry. In this year’s InterLab study the following members of this promotor family where investigated:J23101 (Device 1, strong), J23106 (Device 2, medium) and J23117 (Device 3, weak). As a negative control the TetR repressible promotor (R0040) was used. However, with the positive control we had some issues. Originally the positive control should have been GFP under the control of the J23151 promotor (I20270) but apparently this construct was neither in the InterLab measurement kit (tube was empty) nor in the respective backup well in the distribution (Kit Plate 3, well 8P). We didn´t have the time left to build the positive control from scratch or identify which construct it actually was, but after communication with iGEM HQ we used it anyway.

Results

For the first time we used flow cytometry in order to measure the respective constructs. However, we made some changes to the flow cytometer protocol in order to utilize the know-how for measurements like this already present in our lab. The main changes were that we didn´t use pre- and main cultures and directly used cultures inoculated with the colonies from the construct transformations. Following the incubation, the OD was measured of each culture and an amount of culture was pelleted which would result in an OD of 2 when resuspended in 1 ml PBS. Prior, the measurement the cells where diluted again 1:3 with PBS, filtered through a 100 µm filter (to avoid clocking of the flow cytometer) and then measured with the 530/40(488) channel of the flow cytometer.

Calibration

In order to calibrate the BD Influx Cell Sorter flow cytometer to units of MEFL (molecules of equivalent fluorescence) a sample of SpheroTech RCP-30-5A Rainbow Calibration Particles were used. They are a mixture of different particles with certain numbers of MEFL on them. The measurement of 50000 particles allowed us to connect different MEFL values with their corresponding fluorescence intensity. Table 1 shows fluorescence values for the 8 different MEFL values as well as the conversion value to calculate MEFL from fluorescence after the actual construct measurement:

Mean conversion ratio: 20.08

Measurement of Constructs

After setup of the flow cytometer and calibration of fluorescence and MEFL each of the triplicates of the 5 constructs was measured for its fluorescence. For each sample 50000 events were recorded and the geometric mean of fluorescence (FITC channel  530/40 (488)) was calculated. This fluorescence value is the converted to MEFL using the mean conversion ratio obtained during calibration. Images 1-5 show the point cloud diagrams of fluorescence over side scatter (exemplary for one of the three replicates for each construct) while table 2 shows the calculated results:

Discussion

Even though the construct for the positive control was not the correct one every other result met the expectations. The fluorescence intensity increased with increasing promotor strength while the negative control shows online background fluorescence. However, for device 3 the fluorescence intensity also was really low and comparable with background fluorescence values.
The choice of the flow cytometer as a novel measuring tool seems to be a good decision because it offers distinct values for single particles (cells) which can be linked really well to MELF values obtained during calibration instead of having to use certain reagents to find mean values for particle fluorescence in classic photometer / plate reader approaches.

Protocols

In the absence of an already established flow cytometry protocol we used the approach under “Results” already established in our lab for the actual measurement as well as the preparation steps (transformation, cultivation etc.). For the measurement itself a FITC channel with 530/40(488) filters was used and for calibration the recommended SpheroTech Rainbow Calibration Particles RCP-30-5A.