Team:YiYe-China/Design
Design
Colorectal cancer is one of the most common cancers in the world. The current method for early diagnosis of colorectal cancer remains as a big limitation. Previous scientists identified syndecan-2 (SDC2) and tissue factor pathway inhibitor 2 (TFPI2) were aberrantly methylated in Colorectal Cancer. Quantitative methylation-specific PCR (qMSP) for detecting methylated SDC2 and TFPI2 has been reported as an effective early screening method for colorectal cancer. Toehold switches are a class of RNAs with a hairpin loop that can be unfolded upon binding a trigger RNA for translation initiation of the reporter protein, which plays an important role in clinical diagnoses. In this study, we constructed a toehold plasmid and incubated it in the cell free protein expression system with methylated TFPI2 with a trigger sequence, which offers a non-invasive and visible approach for early detection of colorectal cancer in the future.
Toehold Switch
To be able to detect colorectal cancer polyps as soon as possible before they spread, we decide to use toehold switch which activates gene expression in response to cognate RNAs with arbitrary sequences.
To sum up, a toehold switch is an RNA that encodes gene for protein. Composing of the ribosome binding site and the starting sequence AUG, the toehold switch is in a hairpin structure, but translation of the gene is initially turned off. Toehold switch is designed to detect RNA, and it contains a sequence that is perfectly complementary to the target RNA in the early half of the hairpin. When the target RNA is present, toehold switch recognizes it and binds to it, thus opening the hairpin. Consequently, the initial RBS and AUG are exposed. The reported protein can now be expressed and we could see them glow in color.
Compared to conventional switch, RNA and target RNA are interacted via linear-linear domains, which helps open the switch, thus detecting the cancer polyps.
Fig.1 Design schematics of conventional riboregulators and toehold switches
Benefit: • Less pain than coloscopy • More precise in detecting cancer polyps as the detected protein can glow in color • Much easier to operate and require no additional examination
The role of DNA hypermethylation
If a gene necessary for DNA repair is hypermethylated, resulting in deficient DNA repair, DNA damages will accumulate. Increased DNA damage tends to cause increased errors during DNA synthesis, leading to mutations that can give rise to cancer.
Therefore, patients with cancers have very different sequences, which enables us to detect the cancer cells by using DNA methylation. DNA methylation is a biological process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoter, DNA methylation typically acts to repress gene transcription.
DNA methylation analysis does not require any special handling of tumor specimens and can also be applied with similar efficiency to fresh frozen and formalin fixed paraffin embedded tissue. Also, DNA methylation analysis is not limited to tissue specimens and can be readily extended to almost any bodily fluid.
Parts Design
In this study, we plan to construct a toehold plasmid and incubate it in the cell-free protein expression system with methylated TFPI2 or SDC2 with a trigger sequence, which offers a non-invasive and visible approach for early detection of colorectal cancer in the future.
Fig.2 The parts we designed for CRC dectection.
According to our experimental design, we can judge colorectal cancer based on our experimental results.
Table1 Judgment according to the result of our experiment. 
[1] Green A , Silver P , Collins J , et al. Toehold switches: de-novo-designed regulators of gene expression.[J]. Cell, 2014, 159(4):925-939.
[2] Zhang, Weisong, et al. “SDC2 And TFPI2 Methylation in Stool Samples as an Integrated Biomarker for Early Detection of Colorectal Cancer.” Cancer Management and Research, Dove, 30 Apr. 2021, www.ncbi.nlm.nih.gov/pmc/articles/PMC8096344/.
Contribution
In order to provide useful support and contribution to future iGEM, we have carried out the following two aspects of work.
- Design
- Toehold Switch
- The role of DNA hypermethylation
- Parts Design
- References