Team:UNOTT/InterLab
INTERLAB STUDY:
Introduction
Our team decided to take part in the iGEM interlab study this year. This study is organised by iGEM and is aimed at tackling a prominent issue regarding experimental fluorescent reporter data. At present, experiments using fluorescent reporters are almost incomparable; separate groups often interpret fluorescence measurements in largely different ways, making it hard to properly compare results. To combat this, the interlab study is performed each year, iteratively refining an optimal protocol for fluorescence measurement. Theoretically, this would be followed by anyone and yield comparably robust units of fluorescence, facilitating more meaningful comparisons of data between researchers. The study required us to measure a number of different fluorescent test devices sent to us by iGEM HQ, we followed their protocol and then shared the results we obtain. Teams around the world do the same; collectively, we provide a large set of data illustrating how similar the results were, potentially highlighting any aspects in which the protocol could be improved and, hopefully, showing that this is possible. This is quite an important issue, we felt we should participate and help in any way we could!
Methods
We were given 8 specimens to measure, termed “test devices”. These included a positive (BBa_I20270) and negative(BBa_R0040) control, and 6 different test devices (BBa_J36400, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005).
First we transformed these into DH5α-E. coli cells, then selected two resulting colonies to be prepared for measurement. The two colonies were grown overnight.
But before the experiment could be undertaken, we needed to create calibrate our equipment by performing standard reference measurements, enabling the conversion of our data to absolute values. Firstly, we measured a solution of half LUDOX-S40, half water; fluorescein was then serially diluted by half 10 times and measured to give a standard curve.
After 18 hours of growth, their optical density was measured and a new batch of LB/Chloramphenicol was inoculated to produce an OD of 0.02. These were then grown at 37°C, OD600nm and fluorescence were measured at 0, 2, 4, and 6 hours.
For a full description of the methods, please visit the interlab official study page
Results
LUDOX-S40 and Fluoroscein Reference Measurements
OD600nm and Fluorescence Results
µM Fluorescein/OD600 and Ln µM FITC/OD600 Results
