Team:Fudan/HCC
When the aroma of wine fills the bistro and bard's harp rings jubilant; Or when starlight shines above the earth, declaring the busy day coming to an end; Storybooks are going to be opened; words will come to live again. All sufferings turned to gold, and all whishes became accomplishments. It was a piece of history that us human struggling with cancer. "SwordS", as they called the name.
Hepatocellular carcinoma, HCC is a kind of malignant tumor occurred in liver that has a universal feature—the presence of structural and numeric chromosomal abnormalities indicative of genomic instability. In most cases, it develops from small-cell, high-grade dysplastic nodules in cirrhotic livers. On histologic examination, HCCs range from well-differentiated lesions that reproduce hepatocytes arranged in cords, trabeculae or glandular patterns to poorly differentiated lesions, often composed of large, multinucleate anaplastic giant cells. It may be unifocal, multifocal or diffusely infiltrative in pathologic samples. HCC has a strong propensity for vascular invasion and recapitulates normal liver architecture to varying degrees. In summary, HCC is a serious malignant tumor with high heterogeneity. (Adapt from Robbins Basic Pathology 9th)
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 are ready for experiments through the provided protocol. Generally, plate reader is suggested as the 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.The negative control group shows a high growing comparing to the positive control.
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.
