Before starting cell measurements work, we accomplished three sets of calibration measurements with materials provided by the committee, including an OD600 reference point, a particle standard curve and a fluorenscein standard curve. These tests and data greatly facilitated our following quantitative interlab experiments.
Calibration 1 OD600 reference point and LUDOX
According to the protocol, we performed three calibration experiments (OD600 reference point, particle standard curve and the fluorescence standard curve) before the body part began.
In this experiment, we first uesd LUDOX CL-X provided by the committee to obtain a conversion factor as the intermediate variable between Abs600 and OD600 measurement. The LUDOX solution (45% colloidal silica suspension) is hardly scattering and gives a low absorbance value, making it an ideal material for correct the standard path length of spectrophotometers in different laboratories, which would greatly improve the accuracy of the following volume dependent experiments.
Results shown below.
Fig.1 OD600 reference point
4 replicates are measured respectively to acquire the average Abs600 of LUDOX CL-X solution and dH2O. Reference OD600 is given by the iGEM authority. The ratio of OD600/Abs600 is 3.652.
Calibration 2 Particle Standard Curve
After standardizing the absorbance value, we managed to conclude a conversion factor between Abs600 and the estimated number of cells. In this experiment, we used the silica beads delivered by iGEM committee and made a serial dilution in the 96-well plate according to the protocol. The beads’ size and optical charateristics are similar to cells and its concentration (amount of particles per volume)is known, allowing us to conduct a standard curve of particle concentration in 4 replicates.
Fig.2A Particle Standard Curve
Fig.2B Particle Standard Curve (log scale)
Calibration 3 Fluorescence standard curve
Fluorescence value is an important synthetic biology signal, but it can vary widely in different instruments. As a result, comparing the fluorescence output of test devices by creating a standard fluorescence curve can accurately standardize the amount and fluctuation of GFP fluorescencein in provided kits. In the fluorescence curve measurement, we used the fluorescein powder delivered by committee and made a serial dilution in the 96-well plate according to the protocol. 4 replicates were measured in total.
Fig.3A Fluorescein Standard Curve
Fig.3B Fluorescein Standard Curve(log scale)
Cell measurement
The calibrations experiments above ensured us to complete the cell measurements under the standard conditions. In order to get the valid measurement results, the following tests were all finished with the same settings as we used in calibration measurements.
1)preparation
First of all, we transformed the given plasmids into E.coli K-12 DH5-alpha on the plates of LB medium with chloramphenicol of 25μg/ml in 37℃, and picked 2 colonies of each plate over night, inoculating into 5ml Luria- Bertani liquid medium with 25 μg/mL chloramphenicol, incubating in 37°C at the speed of 220 rpm for 16hrs.
Secondly, the cell cultures were diluted to the concentration of Abs600=0.02 for target start and were transferred to the same LB medium of 12mL in 50mL falcon tubes, at the same incubator above. At the time of 0 and 6 hours of incubation, we took off 500μL into 1.5 ml eppendorf tubes of each colony of 8 devices, and placed them on ice to prevent from further growth.
Last but most importantly, we pipetted 4 replicates samples of each devices, 100μL per well, into a 96-well plate. Samples were laid out according to the plate diagram shown in the protocol.
Fig.4 The arrangement of devices
2)Fluorescence and absorbance reading data
We put the 96-well plates mentioned above into the microplate reader, which had already been calibrated during the previous experiments, and the fluorescence raw readings of eight different devices as well as a blank control at 0h and 6h. Two colonies were taken per device, and each colony had 4 replicates.
Fig.5 Fluorescence_Raw_Readings
Fig.6 Abs600 Raw Readings
Fig.7 uM Fluorescein / OD
Fig.8 Net Fluorescein a.u.
Fig.9 Net Abs 600
Fig.10 MEFL / particle
CFU Measurement
1)Sample Preparation
Before starting measurements, we first transferred plasmids BBa_I20270, Well 2B, to E.coli DH5-alpha as positive control, and the same as plasmids BBa_R0040, Well 2D, as negative control. The two colonies of each device were incubated overnight in LB medium with Cam, at the speed of 220rpm. After 16-hour growth, 25μL was taken from each two colonies and mixed with 175μL blank media into a 96-well plate to measure the OD600. The arrangement is as follows.
Fig.11 CFU Measurement
2)Dilution
Cell cultures were diluted to a target OD600 of 0.1 according to the data above with the formula that (C1)(V1)=(C2)(V2), V2 of which is our final volume of 1000μL.
After dilution, we used another 96-well plate to verify the concentration. 3 replicates 200μL of each sample was pipetted with the arrangement as below. Comparing the absorbance of samples and controls, we could count the accurate cell density.
Now that we managed to measure the accurate density, the colonies were taken a series of graded dilution with the same media above at the following arrangement.
3)CFU Calculation
Then, we separately diluted dilution3, 4 and 5 at the proportion of 1:10 with LB media. As a consequence, the final dilution factor is 8*10-4, 8*10-5 and 8*10-6. The final dilution of 3 devices, 2 colonies of each, were taken 100μL to LB plate, incubated at 37℃ for 18hrs.
After growth, we counted the colonies of each plate and filled the following sheets.
Epilogue
At the end of Interlab work, we would like to thank the iGEM Measurement Committee for providing us with the opportunity to take a part in this study and we are looking forward to the final data by taking all the teams' work into account.
