Team:Fudan/InterLab
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“InterLab” is the short form of “International InterLaboratory Measurement Study in synthetic biology”. In 2017, it’s the fourth Interlab since the start. We believed in the meaning of globally collaboration, been touched by the passion, filled with a scene of mission. In our view, it’s another cornerstone for the international scientific cooperation in synthetic biology besides bio-bricks. Also it’s an opportunity to work with other iGEM teams and improve our skills, so we made a decision to take part in the Interlab.
“History gives us the best thing is its enthusiasm.”- Goethe
For the last three years, Interlab study had made great effort to improve the reliability and standardization of fluorescent measurements, which play an important role in synthetic biology. Through the efforts of Measurement Committee and iGEMers all over the world, a robust measurement procedure for green fluorescent protein (GFP) has been developed successfully. This contributes to worldwide communication and collaboration between laboratories and science workers, establishes a platform to easily exchange, compare and confirm your data with others globally. Data has been collected by the iGEM Measurement Department, meaningful results based on the data have also been published. Thanks to all the pioneers fighting for this, we could now stand at the edge of the world, witness the emerging bright future of synthetic biology.
The 2017 interlab study aims at establishing a GFP measurement protocol based on engineering principles that anyone with a plate reader can use in their lab. The key question is how close can the numbers be when fluorescence is measured all around the world. Thus all teams participated in need to use the same protocol to generate in vivo fluorescence signal and measure the related data including OD600 and fluorescence absorbance.
Using GFP to measure the intracellular expression of specific protein is a basic skill for synthetic biology, so the accuracy and standardization are of vital importance. Different promoters and Ribosomal Binding Sequences were tested in six devices. By measuring the OD600 to show the cell density and fluorescence absorbance to show a total expression, the average strength of promoters in each cell could be calculated.
In order to transfer the relative data, Fluorescein Sodium Salt and LUDOX are used to generate a standardized curve.
8 plasmids (Positive Control, Negative Control, Test Device 1, Test Device 2, Test Device 3, Test Device 4, Test Device 5, and Test Device 6) from Kit Plate 6 or Kit Plate 7 were transformed into E. coli DH5-alpha cells. After transformation and cultivation, the cells were ready for experiments through the provided protocol. Generally, plate reader is the recommended measuring tool, but flow cytometer is also acceptable.
Transformation Protocol:
http://parts.igem.org/Help:Protocols/Transformation
Plate Reader Protocol:
https://2017.igem.org/Competition/InterLab_Study/Plate_Reader
Flow Cytometer Protocol:
https://2017.igem.org/Competition/InterLab_Study/Flow_Cytometry
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OD 600 reference point
Using OD 600 and H2O to generate the conversion factor for the transformation later. The average of OD600 is 0.04725; the correction factor (OD600/ABS600) is 5.6666667
Figure1, OD 600 reference point
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Fluorescein standard curve
Dilution serious of fluorescein were prepared and measured in a 96 well plate. A standard curve is generated to correct the cell based readings to an equivalent fluorescein concentration.
Figure 2: Fluorescein standard curve
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Fluorescein standard curve
Dilution serious of fluorescein were prepared and measured in a 96 well plate. A standard curve is generated to correct the cell based readings to an equivalent fluorescein concentration.
Figure 3: Raw OD600 with background
Show the growing curve of different cells indirectlly. The test devices 2,3,5,6 show a higher growing comparing to devices 1 and 4. Comparing to the positive control, the negative control group shows a high growing.
Figure 4: Fluorescence – background
Test devices 2 and 4 show high fluorescence intensity alone with positive control group. Test devices 1 and 5 show a modest fluorescence intensity, while devices 3 and 6 barely show low fluorescence intensity alone with the negative control group.
Figure 5: Cell measurement
In figure 3, alone with the negative control group, several test devices show a high OD600 value, suggesting a relatively unaffected growth, while some devices like test devices 1 and 4 have a low grow rate. Possible explanation for this is that a high expression of GFP as an unnecessary protein will influence the rate of cell growth. Growth peak probably will show up after h6.
In figure 4, highest fluorescence was obtained from device 2, closely followed by positive control and test device 4. Test device 1 and 5 show a modest fluorescence intensity, while test devices 3 and 6 barely have any fluorescence signal as well as the negative group.
Combined with figure 1, we draw the figure 3, showing the expression of GFP on average.
For test device 1, it had a rather high average expression at the start, but decreased later. This may suggest a constitutive high expression of GFP affects the growth and cell activity, since the total Fl signal was depended on both average expression strength and cell density.
For test device 2, though a high total signal intensity was achieved, the average expression was rather low.
For test device 3 and 6, we found a similar results comparing with the negative group, this may result from rather low expressions close to zero and unaffected growth.
Experiment achieved a rather normal result, showing the robustness of the protocol alone with standardized cells and circuit. The Interlab study do offer a platform for global communication, which is of vital importance in synthetic biology in the future.
