Introduction

"Without absolute units you cannot even say precisely how much shorter you are!"

InterLab Study, iGEM

All of the 2016 iGEM teams were invited and encouraged to participate in the Third International InterLaboratory Measurement Study. We, DTU BioBuilders, were excited to participate in the study, which will aid the establishment of a baseline for replicability of fluorescence measurements and identify likely key sources of error. This study focused specifically on the standardization of the way fluorescent measurements are made, which allows for easier comparisons. iGEM provided the teams with standard protocols whereof we carried out the measurements using the plate reader. In addition, the teams were provided with a pre-constructed Excel data sheet in order to standardize the calculations and presentation of data.

Devices

The 2016 iGEM teams were provided with three test devices, including two controls, a positive and negative control:

• Test Device 1: J23101.B0034.E0040.B0015 in pSB1C3
• Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3
• Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3
• Positive Control Device: I20270 in pSB1C3
• Negative Control Device: R0040 in pSB1C3

Each of the test devices consisted of promotors J23101, J23106 and J23117, respectively, with the first being the strongest. The rest of the test devices was identical consisting of the RBS (B0034), the terminator (B0015) and the GFP (E0040).

The negative control consisted of the pTetR promoter (RR040) with no sequence downstream, and the positive control consisted of the constitutively expressed GFP device (I20270).

All five constructs were already in the pSB1C3 plasmid, which carried chloramphenicol resistance.

Materials and Methods

We used the plate reader protocol provided by iGEM and used the Cytation 5 Cell Imaging Multi-Mode Reader for all our measurements.

Calibration Protocol

OD₆₀₀ Reference Point

100 µL LUDOX provided in the InterLab Measurement Kit was transferred to a Greiner Bio-One 96-well plate in wells A1, B1, C1 and D1. 100 µL demineralized H₂O was added into wells A2, B2, C2 and D2. The absorbance was measured at 600 nm of the eight samples and the data was imported into the provided Excel sheet in the OD₆₀₀ reference point tab.

FITC Fluorescence Standard Curve

The FITC stock tube provided in the InterLab Measurement Kit was spun down to make sure that the pellet was in the bottom of the tube. 1 mL of PBS was added in the tube to make a 2x FITC stock solution (500 µM). The solution was incubated at 42℃ for 4 hours. The stock was then further diluted to 1x FITC solution (250 µM). 200 µl of 1x FITC stock solution was added to A1, B1, C1 and D1. 100 µl PBS was added to wells A2-12, B2-12, C2-C12 and D2-12. 100 µL from A1 was transferred to A2. The new dilution was mixed by pipetting and 100 µL was transferred to A3. This process was repeated until A11 was reached and 100 µL of the final 200 µL in well A11 was discarded. The same process was repeated for rows B, C and D. All of the samples were measured in the 96-well plate and the excitation filter was set to 395 nm and the emission filter to 509 nm. The data were imported into the provided Excel sheet FITC standard curve tab.

Cell Measurement Protocol

The three test devices and the positive and negative control were transformed into competent Escherichia coli DH5Α cells using the provided transformation protocol from iGEM. The transformants were plated on LB with chloramphenicol and incubated at 37℃ overnight.

Next day, two colonies were picked from the plate with the negative control, whereas only one colony was picked from the plates with the test devices and the positive control. The colonies were inoculated in 5 mL Luria-Bertani (LB) medium with chloramphenicol. The cells were incubated at 37℃ and 150 rounds per minute (RPM) until the next day.

The initial OD₆₀₀ of the overnight cultures was measured and important to the "Normalization tab" in the provided Excel sheet. The cultures were diluted to a target OD₆₀₀ of 0.02 in 10 mL LB media with chloramphenicol in 50 mL falcon tubes. 100 µL samples of each solution was transferred to a 1.5 mL Eppendorf tube and placed on ice, while the rest of the solutions were incubated at 37℃ at 150 RPM. 100 µL samples of the each culture were taken and transferred to a 1.5 mL Eppendorf tube and placed on ice at 1, 2, 3, 4, 5, and 6 hours.

At the end, all samples were loaded into a Greiner Bio-One 96 well plate using the same layout as the one suggested by the iGEM plate reader protocol. OD₆₀₀ and fluorescence were measured for each sample. The excitation filter was set to 395 nm and the emission filter to 509 nm when measuring the fluorescence. The data was imported into the "Cell measurement tab" in the provided Excel sheet and sent to measurement@igem.org

Results and Discussion

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Figure 1 shows the OD₆₀₀ measurements of each test device and the positive and negative control. The OD₆₀₀ measurements follow exponential growth.

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Figure 2 shows which device contains the strongest promoter. Negative fluorescence values appears from the fluorescence measurements. We obtained higher fluorescence values in wells only containing media compared to the wells containing the test devices and the postive and negative control. Since the values are negative, it is hard to conclude anything from the results. However, the graph indicates that test device 2 contains the strongest promoter. The results from test device 3 has a lower fluorescence compared to the negative controls, which should not be the case.

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The absolute fluorescence indicate the same results as indicated in Figure 2. It appears that test device 2 has the strongest promoter. However, the values are also negative in this plot and the experiment should be repeated. It is also observed that the absolute fluorescence for each construct decreases over time. This decrease may be due to cells not being able to handle the plasmid.

Conclusion

The InterLab study should be repeated since negative fluorescence values were obtained. However, our results indicate that test device 2 has the strongest promoter.