Team:UIUC Illinois/Interlab Study
Interlab Study
Recognizing the fundamental importance of accurate device characterization and measurement, we contributed to the 2016 iGEM Interlab Measurement Study even though it was not required for our track. The Interlab Study aims to solve a current major challenge in synthetic biology, which is the lack of absolute measures of fluorescence. As fluorescence values are widely used throughout synthetic biology for the characterization of device activity, establishing it as an absolute measure is crucial for the advancement of the field.
In order to achieve this, the iGEM foundation has involved numerous teams worldwide for the past two years in the Interlab Study, and published the results1. Participating teams would perform a series of experiments to grow and measure the same fluorescence constructs using their own labs and equipment, and submit their data to iGEM HQ. Now in its third year running, the InterLab has defined a more stricter set of protocols that all participating teams have to adhere to calibrate their instruments, measure absorbance and fluorescence of the provided constructs, and analyze the collected data. Details of this year’s Interlab Study measurement and analysis protocols can be found at the 2016 iGEM Interlab Study page
Access to the flow cytometer in our lab, housed under the Institute for Genomic Biology, is out-of-bounds to undergraduates, which prevented us from carrying out the Flow Cytometer Protocol. Nonetheless, we were glad that the inclusive nature of the Study allowed us to contribute to the Plate Reader Protocol. IGEM HQ provided us with the constructs and control devices on which we performed the measurement protocol, and a spreadsheet which we populated and submitted with our data.
References
1. Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. [Link]
The constructs and controls supplied by iGEM this year were the same ones as last year, and they are:
Test Device 1: J23101.B0034.E0040.B0015 in pSB1C3
Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3
Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3
Positive Control Device: I20270 in pSB1C3
Negative Control Device: R0040 in pSB1C3
The protocols we used to transform and inoculate the cells needed for this measurement experiment are the same as what we have used for our main project, and can be found on our wiki's Experiments Page.
We used the Thermo Scientific Genesys 10S UV-VIS for absorbance/OD600 measurements, and the Tecan Infinite M1000 Pro for fluorescence measurements.
Our analysis results, using the spreadsheet provided by iGEM, are as follows:
Absorbance (OD600) measured using cuvettes in Thermo Scientific Genesys 10S UV-VIS
The trends in absorbance values of the negative control, and of devices 1 and 3, appear to hover around 0 in the chart above because they were on average about a factor of 100 smaller than those of device 2 and the positive control. All constructs had displayed increasing absorbance values throughout the 6 hour duration of the cell measurement protocol, which agrees with expectations that cell growth is occurring throughout the entire experimental period. This ensures that the increasing trends in the fluorescence measurements can be attributed to the growth of the cells over the duration of the experiment.
Fluorescence in absolute units using 96 well plate in Tecan Infinite M1000 Pro
The fluorescence trends reported from the experiment are in strong agreement with what we would expect in terms of the relative strengths of the promoters (devices 1 to 3), which all belong to the “Anderson Promoter” family created by the 2006 UC Berkeley iGEM team. The "Anderson Promoter” family orders the promoter device numbers according to strength, with BBa_J23119 being the strongest, and BBa_J23100 being the weakest. Device 3 uses BBa_J23117 which is the strongest promoter, followed by device 2 which uses BBa_ J23106, and device 1 which uses BBa_ J23106, the weakest promoter of the three.
