Team:UCLouvain/InterLab
This being our university’s second participation in the iGEM competition, we’ve now decided to take part in the fourth international InterLaboratory Measurement Study in synthetic biology.
This is optional and voluntary, not directly related to our main project (BactaSun) but nonetheless a significant component of the iGEM Bronze Medal criteria.
One major challenge in the field of engineering biology is to standardize measurements in order to compare and share data from different labs across the world using different protocols, reagents, devices, device settings …
The Interlab study is a project run by iGEM’s measurement committee to develop a reliable and repeatable measurement procedure for the green fluorescent protein (GFP) which is one of the most used marker in synthetic biology.
Fluorescence data are usually difficult to compare since they are reported in different units or are processed differently according to various research groups. The project’s objective is therefore to improve current measurement tools by converting data into absolute units.
- 2017 InterLab parts
- Positive control (BBa_I20270)
- 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)
- Competent Escherichia coli K-12 DH5-alpha cell
- Plate reader: Tecan Infinite® 200 PRO
- 96 - wells plate
- InterLab Measurement Kit
First of all the device needs calibrating before measuring fluorescence. The goal is to determine the optimal settings for the plate reader to detect the optical density at 600nm (OD600 reference point). In order to do so we established a correction factor using LUDOX.
Then we proceed to establish a fluorescein fluorescence standard curve using 4 replicates of fluorescein dilution series. This will allow us to convert cell based readings to an equivalent fluorescein concentration and finally into a GFP concentration.
As to the cell culture measurements, controls and devices were transformed into DH5-alpha E.coli strain. Two colonies were picked and grown in LB + chloramphenicol overnight at 37°C. Colonies were diluted to the correct OD600 and (subsequently) samples were then measured every 2 hours for 6 hours.
We used an excitation wavelength of 485nm and an emission wavelength of 530nm to measure the GFP activity.
The whole protocol is available here: PDF.
OD600 reference point
OD600/Abs600 (correcting factor) = 3.125
Fluorescein fluorescence standard curve
Colony 1 cell measurements
