Team:McGill/InterLab
InterLab Study
Calibration #1 : O.D. 600 Reference Point
The purpose of this calibration was to produce a factor to convert from Abs600 measurements to O.D. 600 measurements for Ludox CL-X.
All absorbance values taken at 600 nm were recorded by us using a spectrophotometer while the reference O.D. 600 measurement value (also taken by a spectrophotometer) was supplied by iGEM. Using this data, presented in table 1, we were able to calculate a conversion factor of 5.478 in order to obtain the O.D. 600 values from Abs600 values for use in cell density readings.
Table 1. This table shows the absorbance values taken at 600 nm for both LUDOX CL-X and H2O using a spectrophotometer. The corrected Abs600 measurement was calculated by taking the difference between the mean 600 nm absorbances of LUDOX CL-X and H2O. The reference OD600 value was supplied by iGEM and was used to calculate the ratio of OD600/Abs600 which was then found to be 5.478.
Calibration #2 : Particle Standard Curve
The purpose of this calibration was to create a standard 600 nm absorbance curve of microsphere particle concentration. The reasoning behind using microspheres is that they are a good approximation of the size and volume of cells. The microspheres, given with a known concentration, were supplied by iGEM and were used to create the standard curves seen in figure 1. This curve can then be used to approximate the number of cells in a sample based on its absorbance reading at 600 nm.
Figure 1. The calibration between the number of microspheres and the absorbance at 600 nm. (A) A microsphere particle standard curve generated by using a spectrophotometer to take the abs600 readings of different amounts of microspheres/100 µL. (B) A logarithmic graph of the same standard curve generated by using a spectrophotometer to take the abs600 readings of different amounts of microspheres/100 µL.
Calibration #3 : Fluorescence standard curve - Fluorescein Protocol
The purpose of this calibration was to generate a standard curve of fluorescence for fluorescein concentration in order to be able to convert from cell based readings to an equivalent fluorescein concentration. In addition, this standard curve of fluorescence was generated in order to compare the standard fluorescence output between different participating iGEM teams.
Figure 2. The calibration between fluorescence and fluorescein concentration (µM). (A) A fluorescence standard curve generated by taking the fluorescence measurements and plotting them as a function of fluorescein concentration (µM). (B) A logarithmic graph of the same standard curve generated by taking the fluorescence measurements and plotting them as a function of fluorescein concentration (µM).
Cell Measurement: Fluorescence and O.D. 600
Figure 3. A general schematic of the workflow and procedure of the cell fluorescence and O.D. 600 measurements. Image retrieved from: “IGEM 2018 InterLab Study Protocol.” IGEM 2018, IGEM Foundation, 2018, 2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf.
- Day 1: Escherichia coli DH5α cells were transformed with the plasmids listed below (all in a pSB1C3 backbone)
- BBa_R0040
- BBa_I20270
- BBa_J364000
- BBa_J364001
- BBa_J364002
- BBa_J364007
- BBa_J364008
- BBa_J364009
- Day 2: 2 colonies were picked from each of the transformation plates and were inoculated with 5-10 mL of LB medium and chloramphenicol. The cells were then grown overnight at 37 °C and 220 rpm.
- Day 3: Cell growth, sampling, and assay
- A 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor) was prepared
- The absorbance of these diluted cultures was then taken at 600 nm.
- The cultures were further diluted to a target absorbance at 600 nm of 0.2 in a final volume of 12 mL of LB medium and chloramphenicol in a 50 mL falcon tube (covered with foil to block the light)
- 500 μL samples of the diluted cultures were then taken at 0 hours and added into 1.5 ml eppendorf tubes prior to incubation. The samples were placed on ice.
- The remainder of the cultures were incubated at 37°C and 220 rpm for a time period of 6 hours.
- 500 μL samples of the diluted cultures were then taken at 6 hours and added into 1.5 ml eppendorf tubes prior to incubation. The samples were placed on ice.
- The absorbance at 600 nm as well as the fluorescence of the samples was then measured by using the plate setup shown below in figure 4.
- “IGEM 2018 InterLab Study Protocol.” IGEM 2018, IGEM Foundation, 2018, 2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf.
Figure 4. The general setup of the plates for the measurement of the samples' fluorescence and O.D. 600 readings. For each sample, 100 µL was added to its respective well with 4 replicate samples being produced for each colony. Two plates will be made this way (one at t = 0 hours and the other at t=6 hours). Image retrieved from: “IGEM 2018 InterLab Study Protocol.” IGEM 2018, IGEM Foundation, 2018, 2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf.
Results
Fluorescence per O. D.
Fluorescence per particle
Appendix: Raw Data
Fluorescence Raw Readings
Absorbance at 600 nm Raw Readings
