Team:Montpellier/InterLab
Goal of the study
The International Interlaboratory Measurement Study in Synthetic Biology aims for four years at creating an highly replicable experiment and make it test by numerous labs in the world. This has lead to the robust measurement procedure for the Green Fluorescent Protein (GFP). This protein was chosen because it is one of the most used marker in biology and, as a result, most laboratories are equipped to measure it.
The 5th Interlab Study
The goal of this year study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab.
This year study is to prove whether or not it is possible to reduce variability of GFP measurements by normalyzing to absolute cell count or Colony Forming Unit (CFU) instead of Optical Density (OD).
The Experiments
There are two main part for this year’s interlab study :
- The first is based on the conversion between absorbance of cells to absorbance of a known concentration of beads. With this method we can convert the cells absorbance measured by each lab into a standard “equivalent concentration of beads”.
- The second part is designed for the analysis of CFU. The CFU permits to know the number of live cells coming from a certain volume. Determining the number of CFUs allows to compute a conversion factor between absorbance and CFU.
Link to the Interlab protocol : click here.
Results
We are presenting here the results of iGEM Montpellier team after conducting the protocol for the fifth interlab study.
Before doing the experiment three calibrations are required.
Calibration 1 : OD600 Reference point - LUDOX Protocol :
LUDOX CL-X is used as a single point reference to obtain a conversion factor between the plate reader absorbance at 600nm we are measuring and an absorbance at 600nm a spectrophotometer could measure.
Calibration 2 : Particle Standard Curve - Microsphere Protocol :
This measurement permits us to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells. We diluted silica beads and measured their absorbance with a plate reader.
Calibration 3 : Fluorescence standard curve - Fluorescein Protocol :
The fluorescence recorded by a plate reader is expressed in arbitrary units. This has to be countered by a last calibration which leads to a standard fluorescence curve. For this experiment we used fluorescein which is a cheaper fluorescent protein than GFP.
Cell measurements :
Link to our result tables : download here.
CFU measurements :
Here is the table of our CFU experiment results.
| Number of colonies | Colony Forming Unit (CFU) | ||||||
|---|---|---|---|---|---|---|---|
| Final Dilution Factor | Final Dilution Factor | ||||||
| Plasmid | Triplicate | 8*104 | 8*105 | 8*106 | 8*104 | 8*105 | 8*106 |
| R0040 A | 1 | 640 | 75 | 2 | 5.12*107 | 6.00*107 | 1.60*107 |
| 2 | 680 | 29 | 4 | 5.44*107 | 2.32*107 | 3.20*107 | |
| 3 | 488 | 74 | 0 | 3.90*107 | 5.92*107 | 0 | |
| R0040 B | 1 | 492 | 51 | 10 | 4.08*107 | 8.00*107 | 4.01*107 |
| 2 | 360 | 32 | 3 | 2.88*107 | 2.56*107 | 2.40*107 | |
| 3 | 600 | 82 | 12 | 4.80*107 | 6.56*107 | 9.60*107 | |
| I20270 A | 1 | 864 | 12 | 8 | 6.91*107 | 9.60*107 | 6.40*107 |
| 2 | 768 | 108 | 0 | 6.14*107 | 8.64*107 | 0 | |
| 3 | 692 | 96 | 10 | 5.54*107 | 7.68*107 | 8.00*107 | |
| I20270 B | 1 | 668 | 21 | 0 | 5.34*107 | 1.68*107 | 0 |
| 2 | 892 | 74 | 1 | 7.14*107 | 5.92*107 | 8.00*106 | |
| 3 | 520 | 81 | 2 | 4.16*107 | 6.48*107 | 1.60*107 | |