Team:YiYe-China/Proof Of Concept
Our previous experimental results were the same as we expected and proved our experimental assumptions. In order to improve the simplicity and ease of operation of our detection method, Readout is an issue needed to be considered. Our plan is to extend the diagnostic tools to community hospitals or examination centers closer to patients in remote areas. This avoids the inconvenience of testing the results in larger hospitals and reduces the waiting time for the result. In addition, people can choose to do CRC screening in medical examination centers, which helps to increase the possibility to diagnose tumors at an early stage.
After studying some igem cases from previous years, thus we plan to use in vitro experiments to realize our vision.
1. In vitro experiment
1.1 Cell-free protein expression
We used the cell-free protein expression system to detect the methylation degree of TFPI2 gene in patients with colorectal cancer. As mentioned in the result, we designed the methylated primers of TFPI2 and the non-methylated primers of ACTB as internal controls. The T7 promoter sequence was added to the forward primer, the trigger primer sequence was added to the reverse primer, a new primer was then synthesized respectively. Using normal human genome and simulated colorectal cancer patient genome as templates, we performed PCR amplification with the same number of gene amplification cycles, and then we transfer the PCR amplified product and the Toehold switch plasmid we designed to the cell-free protein expression system and incubated them at 37°C for 3-4 hours for observation. The results show methyl The PCR product amplified by TFPI2 can emit obvious red fluorescence.
Figure1：Functional verification of in vitro protein expression system. tube1：Water; tube2:Toehold plasmid only; tube3: Negative colorectal cancer samples with ACTB non-methylation PCR product; tube4: Negative colorectal cancer samples with TFPI2 methylation specific PCR product; tube5: Positive colorectal cancer samples with ACTB non-methylation specific PCR product; tube 6: Positive colorectal cancer samples with TFPI2 methylation specific PCR product.
The system control group1&2 without PCR products showed no sign of red fluorescence; the internal control group 3 and group 5 with ACTB non-methylation PCR product showed similar brilliant fluorescence, indicating that the two samples have the same sample size; the negative sample containing TFPI2 methylation specific PCR product and toehold plasmids showed no sign of red fluorescence, while the positive sample showed brilliant fluorescence. The result shows that our system works as expected.
1.2 Fluorescence detection
Fluorescence signal was detected and the following data was obtained. ACTB can be used as both quality control and internal reference to normalize the fluorescence value. Through the analysis of the fluorescence value, we can also make further quantitative analyses to improve the accuracy and sensitivity of the product.
2. Dry Lab experiments
Considering that more gradient experiments require more time and samples, as well as a lot of money, we decided to use the modeling method to explore the relationship between the fluorescence signal and the degree of methylation. Through the modeling, we get the following results. Please refer to the detailed modeling process. Our Model page.
The dry lab attempted comparison between direct mcherry expression and that using toehold switch. The result is shown in figure 2, where certain extent of suppression of expression can be observed upon addition of toehold.
Figure 2. Expression level comparison.
Additionally, mcherry concentrations over a specific timespan under different methylation degree are also plotted as shown in figure 3 and 4 . The predicted mcherry concentration gradient shows a non-linear and non-crossing pattern, indicating successful differentiation between different methylation degrees.
3. Conclusion
Both the in vitro experiments and Dry Lab experiment proof our concept.
Proof of Concept
In order to provide useful support and contribution to future iGEM, we have carried out the following two aspects of work.
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1. In vitro experiment
1.1 Cell free protein expression
1.2 Fluorescence detection
- 2. Dry Lab experiment
- 3. Conclusion