Team:Rice/InterLab
Interlab
Is there variability of synthetic biology measurements taken between different labs?
Each year, iGEM conducts an experiment known as the InterLab Study and requests all iGEM teams to contribute data to answer the study's question. Continuing from the InterLab study conducted last year, this year's InterLab study aimed to discover if there is variability in synthetic biology measurements taken between different research labs and whether this variability is significant compared to measurements taken within the same lab.
The Rice iGEM team contributed to this study by running calibrations, preparing samples, and taking measurements in our university's lab according to the protocols provided by iGEM. We tested the following devices provided in the iGEM DNA distribution kits:
Test Devices for iGEM InterLab Study 2018
| Device | Part Number |
|---|---|
| Negative control | BBa_R0040 |
| Positive control | BBa_I20270 |
| Test Device 1 | BBa_J364000 |
| Test Device 2 | BBa_J364001 |
| Test Device 3 | BBa_J364002 |
| Test Device 4 | BBa_J364007 |
| Test Device 5 | BBa_J364008 |
| Test Device 6 | BBa_J364009 |
The test devices were all GFP expressing plasmids that we transformed into E. coli DH5α cells using electrotransformation. Samples cultures of each test device were analyzed in the Tecan Infinite M1000 plate reader in our lab at two time points, hour 0 and hour 6, for fluorescence and absorbance (wavelength = 600 nm). In order to achieve this year's InterLab goal of determining whether lab-to-lab variability of fluorescence measurements can be reduced by incorporating cell counts, our results will be compared with the submission of many other iGEM teams to conduct the InterLab study.
Results
First, our team conducted the three calibration procedures outlined in the protocols. The first was the LUDOX calibration used to convert the absorbance readings from our plate reader to iGEM's OD standards.
LUDOX calibration data
The second calibration involved using silica microspheres to construct a standard curve of absorbance per particle concentration based on a known particle concentration per volume.
Silica bead calibration data
The third calibration produced a standard fluorescence curve using fluorescein to establish a comparison standard for our plate reader.
Fluorescein calibration data
Our results show that at Hour 0, the positive control and the GFP test devices had an average fluorescence per cell count that was greater than that of the negative control as expected.
At Hour 6, the average fluorescence per cell count decreased compared to Hour 0 for all samples but show similar trends between samples.
Discussion
Because the trends in our data is consistent between Hour 0 and 6, we expect that the results we will be contributing to this InterLab study are valid. It is strange that the fluorescence per cell count decreased after six hours as it was expected to increase as the cells were allowed to proliferate.