Team:NTHU Taiwan/InterLab
Interlab
Introduction of Interlab study
We participate in iGEM's fifth International InterLaboratory Measurement Study. This study is organized by iGEM's measurement committee in an effort to establish standardized, reliable and repeatable measurement tools for the iGEM community and the synthetic biology community as a whole. In the InterLab study of this year, our team followed InterLab_2018_Plate_Reader_Protocol to conduct the experiment.
Interlab Experiments
The experiments can be divided into three parts: calibration, cell measurement and counting colony-forming units from the sample.
Calibration 1: OD600 Reference point
LUDOX CL-X (45% colloidal silica suspension) is used as a single point reference to obtain a conversion factor to transform absorbance (Abs600) data from our plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer.
We will get the data for OD600 of the H2O and LUDOX. The corrected Abs600 is calculated by subtracting the H2O reading. To convert measured Abs600 to OD600 is to let Reference OD600 divided by Abs600.
Calibration 2: Particle Standard Curve
We prepare a dilution series of monodisperse silica microspheres and measure the Abs600 in plate reader. The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement allow us to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells.
Calibration 3: Fluorescence standard curve
Absolute fluorescence values cannot be directly compared from one instrument to another. In order to compare fluorescence output of test devices between teams, it is necessary to create a standard fluorescence curve.
2.Cell measurement
First, we transform Escherichia coli DH5α with these following plasmids.
Negative control BBa_R0040 Kit Plate 7 Well 2D
Positive control BBa_I20270 Kit Plate 7 Well 2B
Test Device 1 BBa_J364000 Kit Plate 7 Well 2F
Test Device 2 BBa_J364001 Kit Plate 7 Well 2H
Test Device 3 BBa_J364002 Kit Plate 7 Well 2J
Test Device 4 BBa_J364007 Kit Plate 7 Well 2L
Test Device 5 BBa_J364008 Kit Plate 7 Well 2N
Test Device 6 BBa_J364009 Kit Plate 7 Well 2P
Secondly, two colonies from each plate were picked and inoculated in LB medium containing chloramphenicol overnight for 16-18 hours at 37°C and 220 rpm.
Next, we measure the OD600 and fluorescence of transformed cells according to the protocol after 0, 6 hours. Measurements gave us the following data and calculations which were conducted with the values we obtained from the standard curves and the reference point.
3. Counting colony-forming units (CFUs) from the sample.
This measurement can be used to calibrate OD600 to colony forming unit (CFU) counts, which are directly relatable to the cell concentration of the culture.
We first dilute overnight culture to OD600 = 0.1 in 1mL of LB + Cam media, then prepare a dilution series as instruction. Spread plate for dilution sample and incubate at 37°C overnight and count colonies after 18-20 hours of growth.
