The InterLab studies have been contributing in developing a robust measurement procedure for green fluorescent protein (GFP) over several years now. The goal of this year’s InterLab to identify and correct the sources of systematic variability in synthetic biology measurements of GFP. In order to answer the following research question formulated by the iGEM Measurement Committee: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFU) instead of OD? The following two approaches were used to obtain results for this study:
1. Converting between absorbance of cells to absorbance of a known
concentration of beads.
2. Counting colony-forming units (CFUs) from the sample.
The results of these two approaches were compared to determine how much the results were in accordance with each other to see whether using one (or both) of the methods can help to reduce the lab-to-lab variability in measurements. To get the best comparable results between all the teams performing the InterLab studies, the InterLab Study Protocol was followed explicitly. The first step performed was the transformation of eight strains with plasmids from the iGEM 2018 Distribution Kit. The different strains contained a Negative control, Positive control and six test strains which expressed different levels of GFP. The strains and their corresponding plasmids (parts) used from the Distribution Kit for transformation are shown in table 1. For transformation the iGEM protocol was used.
|Negative control||BBa_R0040||Kit plate 7||Well 2D|
|Positive control||BBa_I20270||Kit plate 7||Well 2B|
|Test Device 1||BBa_J364000||Kit plate 7||Well 2F|
|Test Device 2||BBa_J364001||Kit plate 7||Well 2H|
|Test Device 3||BBa_J364002||Kit plate 7||Well 2J|
|Test Device 4||BBa_J364007||Kit plate 7||Well 2L|
|Test Device 5||BBa_J364008||Kit plate 7||Well 2N|
|Test Device 6||BBa_J364009||Kit plate 7||Well 2P|
2. Experimental Setup
When the transformation of the test devices was confirmed these strains could be used for the two experiments of the InterLab. But first, the three calibration protocols should be completed. The three calibration protocols consisted of measuring an OD600 reference point using the provided LUDOX CL-X (45% colloidal silica suspension), creating a Particle Standard Curve using the provided Microspheres and creating a Fluorescence Standard Curve using the provided Fluorescein. The Particle Standard and the Fluorescence Standard Curve are shown in figure 1 and 2, all measurements were performed in duplo.
After calibration the next step was performing the cell measurement protocol to relate the absorbance and the known concentration of beads (model cells) and to perform the Colony forming units experiment to relate the absorbance and fluorescence to the amount of colonies formed. The results obtained are shown in figure 3 and 4.
With these results and with that the completion of our InterLab studies we hope to have contributed well to the InterLab study of 2018. Working on the InterLab gave us a great start in contributing to the iGEM community. Next to that several core skills were included and resulted in the learning of very useful key skills for later in our project. Even though that the protocol was set up beforehand executing the protocol in detail, to make sure that reproducible results came out of the experiments, was an essential learning point of the InterLab studies. Next to learning some core methods there was still a level of challenging and solution-oriented thinking needed when something, like confirming a successful transformation, did not go right directly.