Team:UPF CRG Barcelona/InterLab
INTERLAB
Introduction
The aim of the Interlab Study is to develop a reliable and repeatable measurement based on cell number, fluorescence, absorbance (optical density) and colony formation units (CFUs). Each year, iGEM teams collaborate in measuring these parameters following the same protocol to obtain a way to have accurate and reliable measurements, which are essential for all sciences, including synthetic biology.
The main part of the Interlab has been always the green fluorescent protein (GFP), one of the biological markers most used in synthetic biology.
The goal of the fifth edition of Interlab is to discover the sources of variability in measurements and be able to correct them, so the measurements taken in different labs will not be variable anymore.
This year question is: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or CFUs instead of OD?
Materials and Methods
Before the starting the experimental part, a plate reader was needed. Due to the lack of plate reader in our laboratory, it was kindly asked to Proteomics and Protein Chemistry Unit (DCEXS-UPF, PRBB) to use their equipment. The plate reader is the Synergy HTX Multimode Reader from Biotek, it can measure both absorbance and fluorescence. It has pathlength correction, which was not disconnected. It has control over temperature and it was set as room temperature (around 24-25ºC). The excitation filter was 485/20 nm and the emission filter 528/20 nm and bottom optics were used. Moreover, the plates were black and flat-bottomed.
Eight plasmids needed to be characterized in DH5-alpha E.coli strain in order to follow the protocol. The strain was obtained by collaboration with BIO-IQS iGEM team. The plasmids are the following: BBa_R0040, BBa_R0040, BBa_I20270, BBa_J3604000, BBA_J364001, BBa_J364002, BBa_J364007, BBa_J364008, BBa_J364009.
The materials used over the protocol are the same ones specified in the iGEM 2018 Interlab Study Protocol.
Results
Calibration
OD600 Reference Point
| Ludox CL-X | H2O | |
|---|---|---|
| Replicate 1 | 0.056 | 0.034 |
| Replicate 2 | 0.056 | 0.034 |
| Replicate 3 | 0.056 | 0.034 |
| Replicate 4 | 0.056 | 0.034 |
| Arithmetic Mean | 0.056 | 0.034 |
| Corrected Abs600 | 0.022 | - |
| Reference OD600 | 0.063 | - |
| OD600/Abs600 | 2.864 | - |
Table 1 | Date from the OD600 Reference Point in calibration 1.
Particle Standard Curve
Figure 1 | Graph showing the Standard curve for the calibration of OD600nm with silica beads.
Figure 2 | Graph showing the Logaritmic Standard curve for the calibration of OD600nm with silica beads.
| Number of Particles | 2.35e8 | 1.18e8 | 5.88e7 | 2.94e7 | 1.47e7 | 7.35e6 | 3.68e6 | 1.84e6 | 9.19e5 | 4.60e5 | 2.30e5 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Mean particles / Abs600 | 7.15e8 | 6.47e8 | 5.04e8 | 4.44e8 | 3.82e8 | 3.27e8 | 2.94e8 | 2.94e8 | 2.04e8 | 1.41e8 | 8.36e7 |
| Mean of med-high levels | - | 4.61e8 | - | - | - | - | - | - | - | - | - |
Table 2 | Particles per Optical density measured.
Fluorescein Standard Curve
This calibration has a R2=0,8547.
Figure 3 | Fluorescence standard curve to calibrate the measurements with fluorescein.
Figure 4 | Logaritmic Fluorescence Standard curve to calibrate the measurements with fluorescein.
| Fluorescein uM | 10.00 | 5.00 | 2.50 | 1.25 | 0.625 | 0.3125 | 0.15625 | 0.078125 | 0.0390625 | 0.0295313 | 0.0097656 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| uM Fluorescein / a.u. | 1.31E-04 | 6,57E-05 | 6.18E-05 | 6.00E-05 | 5.91E-05 | 5.84E-05 | 5.81E-05 | 7.17E-05 | 5.01E-05 | 4.81E-05 | 4.85E-05 |
| Mean uM fluorescein / a.u. | - | 6.10E-05 | - | - | - | - | - | - | - | - | - |
| MEFL / a.u. | - | 3.67E+08 | - | - | - | - | - | - | - | - | - |
Table 3 | Fluorescence in arbitrary units (a.u.) represented by Molecules of Equivalent FLuorescein (MEFL).
Measurement
Fluorescence per optical density
Figure 5 | Fluorescein (uM) related to OD600nm to extrapolate measures of fluorescence with optical density measurements.
Figure 6 | Fluorescence per particle measured at 0 hours and at 6 hours after growing at 37ºC
Colony Forming Units per OD600
Figure 7 | Colony forming units count on each dilution and on each device (postive and negative control)