Team:Nanjing-China/InterLab
Summary
Our team Nanjing-China used E. coli K-12 DH5-alpha to conduct the InterLab Study. The instrument used during our measurements is Tecan Infinite M1000 Pro plate reader which could read both fluorescence and absorbance from the top of the plate. It has variable temperature settings and pathlength correction, which can be disabled. By using this instrument, we have accomplished assignments according to the PLATE READER AND CFU PROTOCOL, and our data has also been accepted.
Problems:
Although we successfully completed the InterLab study ultimately, during the process we also faced some problems which quite puzzled us.
At first, we didn’t understand the purpose of InterLab and were quite confused how to start this project. But fortunately we solved these problems with the assistance from Vinoo Selvarajah, the Director of the Registry and iGEM HQ Representative for the 2018 competition and began this project.
Yet, due to the ignorance of instrumental usage, we were upset by the problem of properly using the plate reader. And our data is not correct because of the improper manipulation. With the help of our secondary PI Peiqing Sun and our advisor Kunlun Li, we finally obtained the correct data and the data were accepted successfully.
During the process of accomplishing the InterLab project, we experienced success and failure, we have also learned plenty of things, such as the manipulation of some instruments, scientific methods and so on.
Details
Calibration Protocols
We used black plates with transparent bottom for the calibration measurements, which had flat-bottomed wells.
Calibration 1: OD600 Reference point - LUDOX Protocol
|
LUDOX CL-X |
H2O |
Replicate 1 |
0.0506 |
0.0372 |
Replicate 2 |
0.050799999 |
0.0353 |
Replicate 3 |
0.0517 |
0.033100002 |
Replicate 4 |
0.050500002 |
0.034600001 |
Arith. Mean |
0.051 |
0.035 |
Corrected Abs600 |
0.016 |
|
Reference OD600 |
0.074 |
|
OD600/Abs600 |
4.669 |
Figure 1. OD600 Reference point
We used LUDOX CL-X as a single point reference to obtain a ratiometric conversion factor to transform our absorbance data into a standard OD600 measurement.
Calibration 2: Particle Standard Curve - Microsphere Protocol
Figure 2. Particle Standard Curve
Figure 3. Particle Standard Curve (log scale)
We prepared a dilution series of monodisperse silica microspheres and measure the Abs600 in our plate reader. The size and optical characteristics of these microspheres were similar to cells, and there was a known amount of particles per volume. This measurement would allow us to construct a standard curve of particle concentration which could be used to convert Abs600 measurements to an estimated number of cells.
Calibration 3: Fluorescence standard curve - Fluorescein Protocol
Figure 4. Fluorescein Standard Curve
Figure 5. Fluorescein Standard Curve (log scale)
We prepared a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in our plate reader. By measuring these in our plate reader, we generated a standard curve of fluorescence for fluorescein concentration. We would be able to use this to convert our cell based readings to an equivalent fluorescein concentration.
Cell measurement protocol
After completing all three of the calibration measurements, we started performing the cell measurements. We use E. coli K-12 DH5-alpha strain, the same plates and volumes that we used in our calibration protocol, as well as the same settings (e.g., filters or excitation and emission wavelengths) that we used in our calibration measurements for the sake of consistence.
Address
Life Science Department
#163 Xianlin Blvd, Qixia District
Nanjing University
Nanjing, Jiangsu Province
P.R. of China
Zip: 210046
