Team:UAlberta/InterLab
Overview
Green Fluorescent Protein (GFP) is a staple in many laboratories as its ability to be fused to proteins of interest, combined with its modulatable fluorescence, provides an easily detectable, and sometimes quantitative, readout that can be used to study different aspects of biological systems. However, the variability in measurement protocols, and the inherent difference between plate reader models, make it difficult to compare fluorescence measurements between different machines, and even between different instances of measurement. The 2017 InterLab study is intended to address this issue by compiling fluorescence measurements from iGEM teams across the world in order to design a reliable measurement protocol which reconciles the discrepancies between different measurement systems. In this investigation, eight devices (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005, BBa_I20270, and BBa_R0040) with different RBS devices (BCDs) were tested.
Team UAlberta’s participation in the InterLab study was not only an exciting technical challenge, but also a great opportunity to collaborate with Team UrbanTundra. Obstacles with the initial transformation of the InterLab devices and the lack of required equipment, among other reasons, encouraged Team UAlberta and Team UrbanTundra to work together, allowing our teams to participate in the InterLab Study.
Materials
- 2017 InterLab Measurement Kit
- E. coli DH5a strain
- Costar Black 96-well Plate, Clear Flat Bottom
- Safire2 Microplate Reader
Methods
Following iGEM requirements, Team UAlberta performed measurements according to these 2017 InterLab Protocols
Results
The InterLab protocols produced data for two colonies of each eight devices, with four replicates. Team UAlberta’s completed data collection document, provided by the iGEM Measurement Committee can be found here. The Safire2 Microplate Reader settings are also included. The processed data for the standard curves and the first replicate of the two colonies of each device is presented below.
Sodium Fluorescein Standard Curve
Figure 1: (A) A Sodium Fluorescein Standard Curve generated by measuring the fluorescence in arbitrary units (AU) as it varies with the sodium fluorescein serial dilutions plotted with a linear vertical axis. (B) A Sodium Fluorescein Standard Curve (Log Scale) linearized by plotting the same measurements on a logarithmic vertical axis.
OD600 Measurements
Figure 2: The optical density at 600nm (OD600) of cultures over six hours calculated with the conversion of absolute absorbance at 600nm by using the ratiometric conversion factor determined in the calibration protocols of the first replicates of (A) colony 1 and (B) colony 2.
Fluorescence Measurements
Figure 3: (A) The fluorescence (AU) of the first replicates of (A) colony 1 and (B) colony 2 over a six hour time period.
Fluorescence Normalized for OD600
Figure 4: The fluorescence of each device normalized for OD600 over the six hour measurement period for (A) colony 1 and (B) colony 2.
Discussion
InterLab Measurements
For both colonies 1 and 2, the majority of the devices increased in OD600 indicating that the cultures grew steadily throughout the six hour measurement period. However, Device 1 exhibited a marginal increase in OD600 at the end of the six hours for both colonies, signifying a lack of growth.
From the fluorescence plots, it is clear that Device 2 obtains the greatest fluorescence at the end of the measurement period for both colonies, while Device 1 and 4 have similar fluorescence levels as the positive control. As expected, the negative control lacks fluorescence, though, Devices 3, 5 and 6 also exhibit low signals.
When the measurements are normalized for OD600, Device 1 appears to have the greatest fluorescence, while Device 3 and 6 have the smallest. It is reasonable to attribute the differences in fluorescence and OD600 to the varying strengths of the different BCDs in each InterLab device as variations in ribosomal recruitment dictate the amount of GFP synthesized and cellular resources available for replication. After further investigation of parts in each InterLab construct, the level of fluorescence is consistent to the relative strengths of the RBS present in the device.
Though all replicates need to be analyzed further, the measurements between first replicates of colony 1 and colony 2 indicate that the prescribed measurement settings and InterLab protocols are effective in ensuring consistent measurements. The assessment of the data from all the iGEM teams participating could serve as further corroboration for Team UAlberta’s results.
UrbanTundra InterLab Collaboration
Despite our frustrations due to challenges presented by the the InterLab protocols, Team UAlberta’s troubles, in fact, led to very robust collaborations with Team UrbanTundra. Our participation in the InterLab study was almost jeopardized as our transformations of E. coli DH5a with the InterLab devices failed multiple times, which depleted our plasmid stock from both Kit Plate 6 and Kit Plate 7. So, in an effort to get these devices into DH5a, we reached out to Team UrbanTundra to ask if we could use their plasmids. With their cooperation, we transformed twice the recommended volume of the InterLab devices and were luckily very successful. In return, we grew cultures of each device and prepped the plasmids in order to create a concentrated stock for Team UAlberta to use for their InterLab Measurements. We also showed Team UrbanTundra how to use the Safire2 and guided them throughout their measurements.
Conclusion
Participating in the InterLab Study was certainly a technical challenge, yet, the opportunity to contribute to developing a standardized fluorescence measurement protocol with our international peers, especially Team UAlberta, was rewarding in its own right. Performing the prescribed protocols with adherence to all the InterLab guidelines yielded consistent and expected results, and with the completed InterLab Google Forms, confirms Team UAlberta’s participation in this InterLab Study.
