Calibration experiments
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.
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. Origin data presented at the bottom of this page.
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. Origin data presented at the bottom of this page.