Failure to repeat and reproduce experiment measurements remains one of the main challenges that hinder the accurate and reliable exchange of data amongst scientists from different laboratories across the world, whether it be due to subtle differences in equipment, material handling, or environmental conditions. Continuing the iGEM foundation’s initiative, iGEM Thessaly along with other iGEM teams, was given the opportunity to participate in the 7th Interlaboratory Study to help combat this problem. We are thankful to the Engineering Committee for including us in the 2023 Interlab study and we hope that our contribution is of significance.

Before starting with the protocols for the 3 experiments, we began by preparing the materials provided in the measurement kit and subsequently performing the Calibration protocol. For the preparation of the stock and working dilutions, the protocol provided by iGEM was followed. Freshly sterilized ddH2O and PBS were used and the light-sensitive solutions were stored in foil-covered eppendorf tubes.
Following the Calibration protocol, a 96-well black plate with a transparent bottom was prepared with serial dilutions of known concentrations of the four calibrants. Freshly sterilized ddH2O and PBS were used. The plate was covered to protect its light-sensitive components during transportation and between measurements.

After the calibration protocol was performed, we set out to perform the 3 experiments. For this purpose, we transformed chemically competent E. coli DH5a cells with the 17 devices, following our standard protocol. In order to optimize our chances of getting colonies from the devices, we used 3 ul (for some transformations 2 or 5 ul of resuspended DNA were used, for more details check the Interlab lab book) of the resuspended DNA to transform 80 ul of competent cells. After the heat-shock transformation, 920 ul of LB broth was added to a final volume of 1 ml per aliquot, and the tubes were placed in a shaking incubator at 37°C and 210 rpm for 1,5 hours. The transformants were streaked on LB agar + Cm plates and then placed for incubation at 37°C O/N.
Once we had colonies for all the devices (listed in Tables 1, 2, 3), we stored the plates in the fridge for a few days, to help with the development of the device’s color, so as to make a correct colony selection. This was suggested to us not only by our advisors, but also by members of the Plant Biotechnology lab of our University. The development of colonies with obvious coloring is clear indication that the bacteria do indeed carry the appropriate cargo and will thus perform as intended for the purposes of the experiments. Even though quite a few of the plates did not develop a color, even after a few days in the fridge, we continued with the protocol and performed the experiments with the transformants we had, after communication with the Interlab Committee.

Experiment 1
Device	Registry Code	Fluorescence Color	Well
Negative Control	BBa_J428100	No color	Plate 1, 13M
Positive control	BBa_I20270	Green	Plate 1, 11A
Test Device 1	BBa_J428112	Green	Plate 1, 15C
Test Device 2	BBa_J428110	Red	Plate 1, 13O
Test Device 3	BBa_J428111	Red	Plate 1, 15A
Test Device 4	BBa_J428101	Red	Plate 1, 13I
Test Device 5	BBa_J428108	Blue and Red	Plate 1, 13A
Test Device 6	BBa_J428106	Green and Blue	Plate 1, 13G

Table 1. Test devices for experiment 1 of iGEM’s 7th Interlaboratory Study 2023.

Experiment 2
Device	Registry Code	Fluorescence Color	Well
Negative Control	BBa_J428100	No color	Plate 1, 13M
Positive control Green	BBa_J428112	Green	Plate 1, 15C
Positive control Red	BBa_J428101	Red	Plate 1, 13I
Test Device 1	BBa_J428106	Green and Blue	Plate 1, 13G
Test Device 2	BBa_J428107	Green and Red	Plate 1, 13E
Test Device 3	BBa_J428109	Blue and Green	Plate 1, 13C
Test Device 4	BBa_J428108	Blue and Red	Plate 1, 13A
Test Device 5	BBa_J428104	Red and Green	Plate 1, 13K

Table 2.Test devices for experiment 2 of iGEM’s 7th Interlaboratory Study 2023.

Experiment 3
Device	Registry Code	Fluorescence Color	Well
Negative Control	BBa_J428100	No color	Plate 1, 13M
Positive control	BBa_I20270	Green	Plate 1, 11A
Test Device 1	BBa_J364000	Green	Plate 1, 11C
Test Device 2	BBa_J364001	Green	Plate 1, 11E
Test Device 3	BBa_J364002	Green	Plate 1, 11G
Test Device 4	BBa_J364007	Green	Plate 1, 11I
Test Device 5	BBa_J364008	Green	Plate 1, 11K
Test Device 6	BBa_J364009	Green	Plate 1, 11M

Table 3.Test devices for experiment 3 of iGEM’s 7th Interlaboratory Study 2023.

Colonies showing appropriate color were marked (when feasible). Duplicate cultures of LB broth with Cm were prepared for each of the devices and incubated O/N (16 hours) at 210 rpm and 37°C. The next morning, the intermediate and final dilutions were prepared in the cold room (4°C) of our lab to ensure the halt of the bacteria growth. For experiments 1 and 2, the first plates were prepared and measured at hour 0, while the final dilutions were returned to the incubator for another 6 hours. The plates were again prepared from the final dilutions on hour 6 and another measurement was taken. For the 3rd experiment, after the first plate was measured, it was returned to the incubator along with the final dilution liquid cultures for 6h. Then, the plate was re-measured after 6 hours and another plate was prepared from the final dilution cultures.

To normalize the results amongst all devices’ measurements for all 3 experiments, we divided the average fluorescence with the average absorbance of each colony for each device. The graphs we produced are depicted below.

The graphs clearly indicate that a significant portion of the devices did not perform as expected. Although we followed the protocol diligently for resuspending the distribution kit devices, performing the heat shock transformation, and conducting the experiments, it seems that there may have been an issue from our perspective. This could be attributed to errors in the handling of laboratory equipment or potentially incorrect selection of colonies during the picking process. The raw data from the experiments can be found below:

Calibration

Experiment 1

Experiment 2

Experiment 3

Problems arose mainly with the transformation of the devices, and the development of colonies carrying the devices. Specifically, we noticed that while we were getting a significant amount of colonies for most of the devices (>15), no fluorescence could be detected during the experiments. This problem persisted even after repeating the transformation process and streaking new plates. During the second round of experiments, we received advice to refrigerate the plates for a few days to enhance the development of the device's color. In most cases, the refrigeration step helped us identify the correct colonies, but unfortunately, it did not resolve the issue for devices in the wells 13A, 13C 11G, and 11M.
Another consistent problem throughout our experiments was our inability to achieve the desired accurate dilution of cultures to reach the final optical density (OD) of 0.02. We followed the protocol precisely, conducting the intermediate and final dilutions in a cold room at 4°C. However, we suspect that this challenge may have been influenced by the condition of our pipettes, the quality of the cuvettes used, and the unreliability of our old spectrophotometer.
Lastly, in line with the findings from the previous year's team, we observed that the Luria Bertani medium itself exhibited fluorescence. This inherent fluorescence posed a challenge when making accurate measurements, leading us to believe that switching to a minimal, non-fluorescent medium like M9 could enhance the quality of our study results.