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 eight constructs (iGEM-kit plate 7) listed below were transformed into chemo-competent DH5α E. coli cells.
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)
For the calibration 100 μL of LUDOX-S40 from the InterLab Measurement Kit was added into the wells A1, B1, C1, and D1. Analogical 100 μL of ddH2O was added to the wells A2, B2, C2, D2. The measurement was performed with a plate reader at an absorbance of 600 nm.
Fluorescein-Fluorescence-Measurement (standard curve)
Measurement of different fluorescein concentrations. excitation wavelength: 485nm; emission wavelength: 528nm
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.
OD600-measurement - growth of E. coli cells.
All cells (with different parts) show the expectable exponential growth.
Fluorescence Measurement of E. coli cells
Test device 2, the positive control and test device 4 show the highest fluorescence after 6 hours followed by test device 5. The remaining test devices do not show any increasing fluorescence and stay on the level of the negative control. (Excitation wavelength: 485nm; emission wavelength: 528nm)