We introduced eight plasmids (2 controls and 6 test devices) to E.coli DH5α by following the protocol and then evaluate the measurement by measuring its OD600. We used TECAN infinite M200 PRO as a plate reader.
"Not only in the synthetic biology, but in the general scientific field, it is definitely important to get the result with high reproducibility and high credibility."
"To achieve this, in iGEM, InterLab Study has been conducted by collecting and comparing the result of GFP measurements with a plate reader."
We introduced eight plasmids from the Distribution Kit to E.Coli DH5α.
We conducted three tests and calibrated the machine.
We measured the OD600 and the fluorescence of the transformed cells.
We measured the Colony Forming Units using two types of transformation cells.
We took the following eight plasmids from the Distribution Kit and introduced them to E.Coli DH5α. Then, we selected two colonies and cultured them for 16 hours.
We conducted the following three measurements to get the calibration values as a preparation for the Cell Measurement.
①Measurment of the LUDOX CL-X solution to obtain a conversation factor to transform the absorbance data into a comparable OD600 measurement.
②ABS600 measument of the dilution series of silica beads to construct a standard curve of particle concentration which can be used to convert Abs 600 measurements to an estimated number of cells.
③Fluorescence measurement of the dilution series of Fluorescein to generate a standard curve of fluorescence for fluorescein concentration. You will be able to use this to convert your cell based readings to an equivalent fluorescein concentration.
Discussion about Calibration
From the Experimental results,Particles / Abs600 = 5.18*10⁸ and MEFL / a.u. = 1.24*10⁹ was found.
We measured the OD600 and the fluorescence of the transformed cells in zero and six hours later. Then we evaluated the measurement data by Calibration values.
Discussion about Cell Mesurement
The results show the changes in optical density (λ = 600 nm) of the transformed cells. They show that the cells grew steadily.
However, when we measured the fluorescence, some values appeared negative or go down after 6-hour culturing. Thus, the Fluorescence/OD and MEFL/Particle differed from the theoretical values.
We diluted both positive and negative controls so that their OD600 became 0.1. Then, we prepared dilution series of them and spread 8.0*10³, 8.0*10⁴, 8.0*10⁵ to the agar medium, counted the number of colonies after 18 hours of cultivation.
Discussion about CFU
We counted the CFU (Colony Forming Unit) and the following figures show the number of colony on each plate. Then, we calculated the CFU / mL in the starting sample when the optical density (λ = 600 nm) is 0.1.