In 2017, the iGEM InterLab Study asked teams to help developing a standardised curve of fluorescence measurements and to measure the relative strength of some unknown devices with different RBSs:
- Device 1: BBa_J364000
- Device 2: BBa_J364001
- Device 3: BBa_J364002
- Device 4: BBa_J364003
- Device 5: BBa_J364004
- Device 6: BBa_J364005
- Positive Control: BBa_ I20270
- Negative Control: BBa_R0040
This involved measuring the fluorescence and OD600 of cultures of E. coli DH5α transformed with each construct to compare the relative expression per cell. By asking all iGEM teams to use the same calibration process for the instruments used for the measurements and comparing the results to a measured standard curve, the relative units of fluorescence can be converted to absolute units. We started our contribution by resuspending the appropriate DNA from the iGEM distribution kit.
OD600 reference point
The experiment was carried out on the 10th July 2017. The first step was to use LUDOX-S40 as a single point reference to obtain a ratiometric conversion factor to transform the absorbance data into a standard OD600 measurement. In order to do this, we used a 96-well plate. 100μl of LUDOX were added into wells A1, B1, C1 and D1 of the 96-well plate. The same amount of H2O was added into wells A2, B2, C2 and D2. The parameters used for the TECAN plate reader were:
- Excitation wavelength: 485nm
- Emission wavelength: 530/30nm
- Gain: 45
The corrected Abs600 was calculated by subtracting the H20 reading. The reference OD600 was defined as measured by the reference spectrophotometer (provided by iGEM HQ). The correction factor to convert measured Abs600 to OD600 was hence the Reference OD600 divided by Abs600. In order to maintain consistency of the results, we used the same plates and volumes for the subsequent steps.
Fluorescein standard curve
In this part of the InterLab study, we prepared a dilution series in four replicates in a 96-well plate. By measuring it using the TECAN, we managed to generate a standard curve of fluorescence for fluorescein concentrations, which enabled us to correct the fluorescence measured for the different constructs in E. colito an equivalent fluorescein concentration in the next steps of the InterLab study. As the aim was to make the process as accurate and precise as possible, we programmed an Eppendorf epmotion robot to perform the dilutions for us. We began with spinning down the fluorescein stock tube in a centrifuge to make sure that the pellet was at the bottom. Then we prepared 2x fluorescein stock solution (100μM) by resuspending fluorescein in 1mL of 1xPBS. We pipetted the solution up and down so that the fluorescein was properly dissolved. Finally, we diluted the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and obtain the resulting 50μM concentration of the stock solution. Serial dilutions were performed in a 96-well plate, as mentioned, across columns 1-11. The initial step was to place the fluorescein stock in row A, column 1, and then an equal volume of 1xPBS in columns 2 to 12. Starting from column 1, 100μl was transferred consecutively from column to column until column 11 was reached. Column 12 contained pure PBS.The dilution series was repeated for rows B,C and D. Fluorescence of all samples was measured with TECAN using the same parameters as previously. The data was recorded in a special Excel sheet provided by iGEM.
In order to perform cell measurements using the TECAN, we started by transforming our competent E.coli DH5α cells with each of the seven devices provided by iGEM. They were spread on agar plates containing Chloramphenicol and left to grow overnight in an incubator set to 37 degrees Celsius. The following day we picked colonies from each of the plates, inoculated them using 5mL LB medium with 5μL Chloramphenicol and left them to grow over night at 37 degrees Celsius in a shaking incubator set to 220rpm. To have a backup in case of any mistakes or misunderstandings, we made glycerol stocks of the overnights. We did this by adding 500μL of liquid overnights to 500μL of glycerol and placing them immediately in a -80 degrees Celsius freezer where they could be stored safely. In the afternoon we prepared new overnight culture from plates, using the same procedure as last time, to conduct the required measurements the following day.
We started the next day with measuring the OD600 of each of the overnight cultures. The values allowed us to measure how much additional LB we needed to add to dilute the cultures to a target OD600 of 0.02 in 12mL LB medium with Chloramphenicol in 50mL falcon tubes covered with foil to block light. Our measurements and calculations were as follows:
|Device||OD||LB to be added (µl)|
The cultures were then incubated at 37 degrees Celsius and 220rpm. 500μL samples of the cultures were removed at 0, 2, 4 and 6 hours of incubation. Every set of 16 samples was placed on ice and left there until the last samples were taken after 6 hours. The samples were then transferred into a 96-well plate according to the layout specified in the protocol provided by iGEM HQ. The OD600 and fluorescence were measured at the end of the day, using the same settings as for previous experiments and the data was recorded in a special Excel sheet provided by iGEM.