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.
For all plate reading measurements, we used the Tecan Infinite M1000 Pro. For all curvette measurements, we used the Thermo Scientific Genesys 10S UV-VIS. All initial OD values that were required before starting the plate reader measurements were obtained using curvettes.
The following plots show the trends in absorbance and fluorescence taken over the course of the 6 hour Plate Reader Protocol:
The increasing trend in absorbance values indicate that there was growth in all devices (except for Device 1), including the controls, as expected throughout the entire experimental period. A possible reason for the lack of growth of Device 1 could be that the cells transformed with Device 1 were not inoculated well.
The “Anderson Promoter” family orders the promoter device numbers according to strength. In the context of the Interlab Study, this means that promoter BBa_J23101, in Device 1, is the strongest promoter, while promoter BBa_J23117, in Device 3, is the weakest weakest. Thus, we would expect the fluorescence levels for Device 1 to be the highest among all the devices, followed by Devices 2 and 3. The results for fluorescence are plotted here against a log scale for clarity in comparison across all levels among the devices involved. We see that the results are not the same as what we would expect from our knowledge of the relative strength of the promoters. Given the small number of replicates in the study, it is not possible to draw any accurate conclusions, and repeats will need to be done as part of future work.
