Team:DTU-Denmark/molecular interlab
InterLab
This year’s iGEM InterLab study aim to establish common, comparable units of measurement of fluorescence, which is one of the biggest challenges in synthetic biology.
Introduction
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 carries 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 component 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 LB medium with chloramphenicol. The cells were incubated at 37℃ and 150 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
The above graph shows the OD600 measurements of each test device and the positive and negative control. The OD600 measurements follow exponential growth.
