Team:Paris Bettencourt/InterLab
InterLab
Introduction
Our iGEM Paris Bettencourt 2018 is an enthusiastic group of scientist who wanted to participate in the Interlab study.
This year, the aim was to globally improve the measurement tools for synthetic biology : allowing reliable and reproducible measurements. To do so, teams world-wide participated in a study to follow the same measurement experimental protocol and share their results, such that it permitted comparing and discover the source of variable results.
The objective- to devise a “perfect protocol” to minimise lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of using the standard OD600.
RECOMMENDED PROTOCOL FOLLOWED BY EACH TEAM:
Step 1: Calibrations for fluorescence measurements using LUDOX CL-X, silica beads and fluorescein.
WITH LUDOX CL-X:
LUDOX CL-X is used as a reference to transform the plate reader absorbance data into a comparable spectrophotometer OD600 measurement.
4 replicates of a defined volume of LUDOX CL-X and double-distilled water samples were measured absorbance at 600 nm with the available TECAN plate reader and the outputs were recorded.
WITH SILICA BEADS :
The silica beads provided by iGEM represent artificial Escherichia coli cells, thus, we tested their light scattering pattern. A serial dilution of the silica beads sample in ddH2O, was used to measure the absorbance.
WITH FLUORESCEIN:
Fluorescence by each instrument reports in arbitrary units, making comparisons of values difficult from different instruments to instruments. The study aimed to create a standard fluorescence curve using fluorescein has the same excitation and emission properties as GFP.
Serial dilution of fluorescein in four replicates followed by fluorescence measurement in a 96 well plate reader. With the data, a standard curve of fluorescence was generated using the our in-house reader and equivalent fluorescein concentration.
Step 2: Transformation of competent cells with plasmids and determination of colony-forming units (cfu)
E. coli DH5ɑ competent cells were transformed with 8 differents plasmids. Colonies were selected and grown with the appropriate antibiotic overnight. Fluorescence before and after 6 hours incubation are measured in 4 replicates. Then, the OD600 was calibrated to cfu, serial dilutions of the cell culture in triplicates (with previous positive and negative controls) were performed until a fixed OD600 of 0.1. The cell culture were then plated on and the cfu was measured.
The results obtained have been submitted to the Measurement Committee, to help them generate a system to standardise the protocols across the globe and finally reduce the variability lab-to-lab.
SUGGESTIONS FOR FUTURE INTERLAB STUDIES
Our collaboration with GIFU iGEM from Japan was quite successful and thus, for the future, we propose to an inter lab-study involving the usage of cell-free systems as they are reliable when optimized and give reproducible results. Cell-free systems are the future of protein expression and such an inter lab study would benefit the future iGEMERs.
paris-bettencourt-2018@cri-paris.org 
