IGEM Evry Paris-Saclay

InterLab

Overview

In order to get trustworthy data, there is an important need for normalization. Indeed, different manipulators on the same experiment can introduce bias into the results. There is a recurrent need to assess this variability and to be able to efficiently analyze and compare data. But, nowadays, we have to turn ourselves to relative activity comparisons, complicating the interpretation of our results and the collaboration between different laboratories.

The iGEM InterLab Study aims to correct this by creating a standardized procedure that allows a direct reading using absolute units for the measurement GFP's fluorescence. It should, therefore, allow the performance of reliable and replicable measurements that are a key element for the improving of engineering biology. The study will be conducted by a large number of iGEM teams around the world and the variability of the results will be analyzed.

It is with a great pride that the iGEM Evry Paris-Saclay team participated in the fourth InterLab, the biggest study in that field, which will certainly allow synthetic biology to develop.

Detailed protocol of the 2017 InterLab Study can be found here.

You can see our Raw data here.

Materials

To perform the required experiments, we used the plate reader CLARIOstar^® (BMG LABTECH) lent by the abSYNTH platform of Genopole at the institute of Systems and Synthetic Biology in Evry, France. Measurements were conducted following the provided protocol. The fluorescence has been measured by the camera above the plate with a 90% gain. The 96 well plate we used are the COSTAR^® 3603 from Corning Inc.

Preliminary Tests

Calibration

This step aims to correlate A_600nm and OD_600nm by calculating a ratiometric conversion factor. This basic experiment consists of the measurement of the absorbance at 600 nm of 8 samples. It uses for the first four samples 1 mL of LUDOX (provided in the InterLab kit) and H₂0 for the four others.

Standard Curve

Using the fluorescein (1X in PBS) provided in the InterLab kit, we made a serial dilution and thus dispatch fluorescein at the right concentration in the wells of the 96-well plate. By creating this curve, we can determine a ratio between a fluorescence measurement and the fluorescein concentration.

We observed a linear curve between the fluorescence value and the fluorescein concentration indicating a correlation between the two. (Figure 1).

Figure 1. Standard curve of fluorescence intensity as a function of Fluorescein concentration (µM) — **Figure 1.** Standard curve of fluorescence intensity as a function of Fluorescein concentration (µM).

Cell Measurement

The cell measurement has been performed after an incubation at 37°C in LB (Luria Bertani) media supplemented with Chloramphenicol (35 µg/mL working concentration). Cells were transformed Escherichia coli K-12 DH5α carrying (except for the controls) one of the six following devices:

Test Device 1: BBa_J364000 (BBa_J23101 + BBa_JI13504)
Test Device 2: BBa_J364001 (BBa_J23106 + BBa_I13504)
Test Device 3: BBa_J364002 (BBa_J23117 + BBa_I13504)
Test Device 4: BBa_J364003 (BBa_J23101 + BBa_J364100 + BBa_E0040 + BBa_B0015)
Test Device 5: BBa_J364004 (BBa_J23106 + BBa_J364100 + BBa_E0040 + BBa_B0015)
Test Device 6: BBa_J364005 (BBa_J23117 + BBa_J364100 + BBa_E0040 + BBa_B0015)

As negative control we used the iGEM construction BBa_R0040 and as positive control BBa_I20270.

Method

The procedure we used is schematized in figures 2 and 3.

Figure 2. Schematic representation of the cell preparation for the measurements — **Figure 2.** Schematic representation of the cell preparation for the measurements

Figure 3. 96-well plate plan — **Figure 3.** 96-well plate plan.

Results

Data showed on the graphs (Figures 4 and 5) are the average of the mean of four replicates for each of the two colonies.

Cell Density Measurement

According to our cell density measurements data presented in Figure 4, every device presents the same pattern with an increase of cell density over time that is consistent between 2 and 6 hours but a little bit stronger during the 2 initial hours.

The blank, a LB media supplemented with chloramphenicol, doesn't show an increase of absorbance related to cell growth.

Figure 4. Growth curves of E. coli cells bearing either one of the six tested devices or the positive and negative controls. — **Figure 4.** Growth curves of *E. coli* cells bearing either one of the six tested devices or the positive and negative controls.

Fluorescence

According to our fluorescent measurements data presented in Figure 5, the devices may be classified in two groups of different fluorescence intensity with devices n°2 and n°6 exhibiting a stronger fluorescence than the devices n°3, n°4 and n°5.

The fluorescence of devices n°2 and n°6 appears constant during the four prime hours and followed by an increase of the fluorescence for the rest of the measurement.

The device n°1 show a very high fluorescence intensity as early as t=0 and doesn't seem to present any significant variation during time. We hypothesize that a saturation effect prevents the fluorescence to increase any more, maybe due to the plate reader we used.

We can observe that the negative control showed an increasing fluorescence after 4h even if the value stayed below the fluorescence of the other devices. We can argue that the negative control probably suffered from contamination, which could explain an increase of cell density and fluorescence.

Figure 5. Fluorescence measurement of E. coli cells bearing either one of the 6 tested devices or the positive and negative controls. — **Figure 5.** Fluorescence measurement of *E. coli* cells bearing either one of the 6 tested devices or the positive and negative controls.

Team:Evry Paris-Saclay/InterLab