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Revision as of 21:26, 29 October 2017
Interlab Study
Introduction
Reproducibility of an experiment and reliable measurement results are important parts of synthetic biology but it is very complicated to find a significant number of laboratories that are able to work on a identical protocol to produce comparable results.
Being in many places in the world iGEM seems to be the perfect platform to compare results of one experiment in many different laboratories.
iGEM Stuttgart also took part at the fourth InterLab Measurement Study in 2017.
The aim of this years study was to measure GFP fluorescence of 6 different test devices (+negative and positive control).
The following eight constructs (iGEM-kit plate 7) were transformed into chemo-competent DH5α E.coli cells:
| Positive Control (BBa_120270) |
| Negative Control (BBa_R0040) |
| Test Device 1 (BBa_J364000) |
| Test Device 2 (BBa_J364001) |
| Test Device 3 (BBa_J364002) |
| Test Device 4 (BBa_J364003) |
| Test Device 5 (BBa_J364004) |
| Test Device 6 (BBa_J364005) |
First step of the experiment is the calibration of the available plate reader with LUDOX SH40 and the generation of a standard curve with fluorescein (both provided in the iGEM-Measurement Kit). The next step is the transformation of the mentioned plasmids into DH5alpha E.coli cells followed by the procedure explained in the picture below. Results of the measurements of OD600 and fluorescence after 0, 2, 4 and 6 hours will be compared and used to establish a precise GFP measurement protocol. We used a BioTek Synergy 2 plate reader and clear 96-well plates for both measurements. You can find the protocol, the detailed description of the experiment and information about the InterLab Study itself here.
Results and Discussion
LUDOX-OD600 Measurement (calibration)
HIER TEXT REINKOPIEREN
| LUDOX-HS40 | H2O | |
|---|---|---|
| Replicate 1 | 0,04 | 0,026 |
| Replicate 2 | 0,041 | 0,027 |
| Replicate 3 | 0,043 | 0,026 |
| Replicate 4 | 0,04 | 0,028 |
| Arith. Mean | 0,041 | |
| Corrected Abs600 | 0,0143 | |
| Reference OD600 | 0,043 | |
| OD600/Abs600 | 2,98 |
Fluorescein-Fluorescence-Measurement (standard curve)
Measurement of different fluorescein concentrations. excitation wavelength: 485nm; emission wavelength: 528nm
Transformation
We had at least two colonies of every test device except for test device 1 (transformation not successful). All of the following measurements are without test device 1. Agar-plates with colonies after transformation.
Cell Measurement
OD600-measurement - growth of E.coli cells
Fluorescence Measurement of E.coli cells
Excitation wavelength: 485nm; emission wavelength: 528nm
