Team:ULaval/InterLab

InterLab

Overview

The objective of this year’s interlab is to test a more direct method of fluorescence measurements than those currently used in an effort to achieve better reproducibility in life science experiments. By computing the results of absorbance and fluorescence obtained by hundreds of teams around the world, this study will help identify and correct the sources of systematic variability in measurements. The absorbance and fluorescence measurements were performed with a plate reader which was kindly lent to us by the Department of Biochemistry, Microbiology and Bioinformatics at Laval University. As we also had the chance to be able to access a flow cytometer as well as SpheroTech calibration beads, our team also participated in the cytometry measurements experiment. The cytometer was kindly lent to us by the Landry laboratory (Laval University) while the beads were kindly given to us by the Centre Hospitalier de l’Université Laval (CHUL). The iGEM ULaval team will be pleased to communicate their results upon request: igem@bcm.ulaval.ca

For detailed protocols and more information about the InterLaboratory Measurement Study, please visit the 2018 iGEM interlab page or see the PDF version protocols for the plate reader and flow cytometry.

Calibration

For the calibration, three different protocols were used: an OD₆₀₀ reference point, a particle standard curve, and a fluorescence standard curve. In order to produce valid results, those measurements used the same plates, volumes, and settings than the cell-based assays.

Calibration 1:​ OD_​600 Reference point - LUDOX Protocol

The LUDOX protocol (plate reader, page 2) was used to transform Abs₆₀₀ measurements taken from a plate reader into comparable OD₆₀₀ measurements. The arithmetic means for the absorbance of LUDOX CL-X and water of the four replicates were used to calculate the corrected Abs₆₀₀ (value of 0.045). By using an OD₆₀₀ reference of 0.063, we found a conversion factor of 1.39.

Figure 1. Absorbance measures for each replicate and their arithmetic means.

Calibration 2:​ Particle Standard Curve

A particle standard curve was obtained by plotting the Abs₆₀₀ against several concentrations of monodisperse silica microspheres. The data was analyzed by performing a linear regression analysis.

Figure 2. A₆₀₀ in function
of particle concentration.

Figure 3. Mean Abs₆₀₀ in function
of particle concentration on a logarithmic scale.

Calibration 3:​ Fluorescence Standard Curve

Linear fluorescence standard curves were constructed by plotting the fluorescence intensities against several concentrations of monodisperse silica microspheres. The data was analyzed by performing a linear regression analysis.

Figure 4. Fluorescence intensity in function of fluorescein concentration.

Figure 5. Mean Fluorescence in function of fluorescein concentration on a logarithmic scale.

Cell Measurement

For the cell measurements, a strain of Escherichia coli K-12 DH5-alpha has been used by all the iGEM teams.
Bacterial cells were transformed with the eight following devices:

Devices

Device	Part Number
Negative Control	BBa_R0040
Positive Control	BBa_I20270
Device 1	BBa_J364000
Device 2	BBa_J364001
Device 3	BBa_J364002
Device 4	BBa_J364007
Device 5	BBa_J364008
Device 6	BBa_J364009

Two colonies from each transformation were picked up and incubated overnight in 10mL LB medium + Chloramphenicol at 37°C and 220 RPM. Abs₆₀₀ and fluorescence were measured after 0 and 6 hours of incubation.

The Equation 1 presents the mean of the ratio between net fluorescence and net Abs₆₀₀ multiplied by the ratio of the two unit scaling factor found during Calibration 2 and 3 (i.e. for each replicate).

Equation 1.

The Equation 2 presents the mean of the ration between net fluorescence and net Abs₆₀₀ multiplied by the ratio of the two unit scaling factor found during Calibration 1 and 3 (i.e. for each replicate).

Equation 2.


Table 1. Unit scaling factors calculated at each calibration.

Figure 6. Calculated means for the fluorescein/particle at t=0h and t=6h.

Figure 7. Calculated means (n=5) for the fluorescein/OD at t=0h and t=6h.

Colony Forming Units per 0.1 OD₆₀₀ E. coli cultures

This protocol used both the Negative (BBa_R0040) and Positive Control (BBa_I20270) cultivated during the Cell Measurement. Those for sampling (A1-A2: Positive Control (t=0 and 6h); B1-B2: Negative Control (t=0 and 6h)) were diluted to reach an OD₆₀₀ between 0.097 and 0.239 with the LB+Cam solution. For each culture, triplicates of 200μL were prepared (receptively I, II and III). For all the triplicates, serial dilutions were made and, as a result, three dilutions were selected for each replicate; Dilution 3 (8E-3), Dilution 4 (8E-4) and Dilution 5 (8E-5). These cell cultures were incubated overnight before calculating the number of colonies for each plate and multiplying those number by the Final Dilution Factor (8E4, 8E5 and 8E6, receptively) to obtain the colony forming units (CFU) per 1mL of an OD₆₀₀ of 0.1.

Table 2. Numbers of colonies and CFU per 1mL of Positive (A1-A2) and Negative (B1-B2) Control of an OD₆₀₀ of 0.1 in function of different dilution factors (Dilution 3: 8E4, Dilution 4: 8E5 and Dilution 5: 8E6).