Team:TU Darmstadt/InterLab
Contents
Interlab Study
Since iGEM is an international competition with many teams from all over the world, it is inevitably coping with the problem of unreproducible scientific measurement results. Multiple teams are measuring fluorescence differently, in different units and are evaluating the results non-identically. Therefore, the InterLab Study aims to develop a standardized protocol for repeatable fluorescence measurements of GFP. The study should be carried out and examined in different laboratories all over the world. Our team followed the standardized procedure guiding us through transformation, inoculation and measurement process. For more information: Fifth International InterLab Measurement Study Following below you can find our results.
Calibration
OD600 Calibration
Several measurements of LUDOX-S40 and H2O were spectrometric analyzed below.
| LUDOX CL-X | H2O | |
|---|---|---|
| Replicate 1 | 0.0525 | 0.0355 |
| Replicate 2 | 0.0526 | 0.0351 |
| Replicate 3 | 0.0529 | 0.0373 |
| Replicate 4 | 0.0528 | 0.0352 |
| Arithmetic Mean | 0.0528 | 0.0358 |
| Corrected Absorbance 600 | 0.0169 | |
| Reference OD600 | 0.0630 | |
| Correction Factor | 3.7223 |
Table 1. Absorbance measurement of LUDOX-S40 and H2O of four different replicates.
Defining the correction factor for upcoming experiments allows the conversion of Absorbance 600 from a plate reader into OD600.
Figure 1. Data exported from table 1 in beam chart.
Particle Standard Curve
Table 2.
Figure 2.
Figure 3.
Fluorescein Standard Curve
The fluorescence of a dilution series, fluorescein in four replicates, were measured in standard modes in our tecan plate reader (infinite 200Pro).
Table 3. Fluorescence measurement of fluorescein in eleven dilutions and one control containing PBS. Measurement settings for the plate reader: Excitation 485 nm, Emission 530 nm, 50 gains and 20 flashes.
Generated standard curve of fluorescence for different fluorescein concentrations can be found below.
Figure 4. Calibration curve of fluorescein: fluorescence measurement of different concentrations (µM).
Figure 5. Calibration curve of fluorescein (log scale): fluorescence measurement of different concentrations (µM).
Cell Measurement
Fluorescence and absorbance was measured from six different devices, positive and negative control which are built in pSB1C3 containing chloramphenicol resistance. Samples were taken after 0 and 6 hours. The arithmetic mean from two colonies was calculated and can be found below.