Team:BGU Israel/InterLab
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InterLab
Our team chose to participate in the Fifth International InterLab Measurement Study in synthetic biology.
The goal of the iGEM InterLab 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.
In this study we want to reduce lab-to-lab variability in fluorescence measurements by normalizing it to absolute cell count or colony-forming units (CFUs) instead of OD.
The fluorescence in the bacteria in our experiments occurs due to the presence of a gene that encodes a green fluorescent protein: GFP.
In cell and molecular biology, the GFP gene is frequently used as an expression reporter protein. Reporter genes are often used as an indication that a certain gene has been internalized or expressed in cells or organisms.
Equipment
All of our measurements were taken in a plate reader that can read both absorbance and fluorescence (Neoteck-Tecan-INFINITE M100) without a pathlength correction. The temperature was set to 25̊C, under shaking with a duration of 3 sec and an amplitude of 2 mm. We went through a tutorial and learned how to operate the machine.
In addition, we used the following reagents (Partially supplied by iGEM):
- 1.0 ml LUDOX CL-X
- 150 μL Silica Bead (microsphere suspension)
- Fluorescein (powder, in amber tube)
- iGEM Parts Distribution Kit Plates
- 1 x PBS (phosphate buffered saline, pH 7.4 - 7.6)
- ddH2O
- Competent cells (Escherichia coli DH5α)
- LB (Luria Bertani) media
- Chloramphenicol
- 96 well plates, black with clear flat bottom
Safety procedures
As part of an ongoing work in a biological laboratory, safety gear was used to work with bacteria. The work was done with closed shoes, long pants, gloves and lab coats.
In order to maintain a clean environment, and sterility between the samples the work was performed next to a flame, and all the disposable parts were thrown after a single use into a biological waste bin.
Before and after work, work surfaces were sterilized with 70% alcohol.
Protocols
Calibration 1: OD600 Reference point - LUDOX Protocol:
100 μL LUDOX were added into wells A1, B1, C1, D1.
100 μLof ddH2O were added into wells A2, B2, C2, D2
| LUDOX CL-X | H2O | |
|---|---|---|
| Replicate 1 | 0.0561 | 0.029 |
| Replicate 2 | 0.0522 | 0.0266 |
| Replicate 3 | 0.0554 | 0.033 |
| Replicate 4 | 0.0534 | 0.0292 |
| Arith. Mean | 0.054 | 0.029 |
| Corrected Abs600 | 0.025 | |
| Reference OD600 | 0.063 | |
| OD600/Abs600 | 2.538 |
Table 1: OD600/Abs600 measurements.
The correction factor is 2.538.
Calibration 2: Particle Standard Curve - Microsphere Protocol
- The Silica Beads from the InterLab test kit were vigorously vortexed for 30 seconds.
- 96 μL microspheres were pipetted into a 1.5 mL Eppendorf tubes
- 904 μL of ddH2O were added to the microspheres
- The Microsphere Stock Solution was vortexed well
- 200 μL of Microsphere Stock Solution were transferred into each well in column 1
- 100 μL of ddH2O were added into each well (2-12) in the corresponding row
- Serial dilution by transfer of 100 μL from column to column with good mixing
- Absorbance measurements of all the samples in the plate reader in 600 nm
Figure 1: Particle Standard Curve
Figure 2: Particle Standard Curve log scale
We got a linear correlation between the Abs600 and the amount of particle.