Improvement

The introduction of our experiment:

We have established a toehold switch that combines with a fluorescent protein, mCherry, on its tail to detect methylated TFPI2 for early screening of non-invasive diagnosis of CRC (https://2021.igem.org/Team:YiYe-China). It is reported that SDC2 methylation and TFPI2 methylation are highly sensitive to CRC(DOI:10.2147/cmar.s375358) . In order to further improve the detection accuracy of CRC compared to last year, we combined the detection of methylated SDC2 with methylated TFPI2 this year. We constructed a toehold switch consisting of the plasmids of double-toehold pSB1C3 (BBa_K4218003) and double-trigger pCOLADuetTM-1, and then we have a functional verification for the plasmids in the BL21 cells. Finally, we detected methylation SDC2 gene and methylated TFPI2 through the expression of fluorescent protein in cell-free protein expression system.

1.Construction of the plasmids

Considering two different plasmids have been co-transformed into E. coli to verify the functions, two plasmids need to have different origin of replication (ori) (Figure 1). Therefore, we inserted the Toehold sequence and mcherry sequence into pSB1C3 and cloned the trigger sequence into pCOLADuetTM-1. We used SpeI / XbaI enzyme to digest PSB1C3 vector and NdeI/XhoI enzyme to digest pCOLADuetTM-1 vector (Figure 2). The synthesis of double Toehold sequence and TFPI2 and SDC2 trigger sequence were linked to the enzyme digestion of pSB1C3 vector (double toehold pSB1C3) and pCOLADuetTM-1 vector (double trigger pCOLADuetTM-1). Then the ligation products were transformed into DH5alpha bacteria. Positive clones were selected on LB plate containing chloramphenicol and kana antibiotics, respectively. After overnight incubator at 37 ℃, the positive clones were picked out and amplified in LB tube for 6 hours, and then 100 μl bacteria solution was extracted and sent to Tsingke Biology Company for sequencing (Figure 3).

Figure 1. The final structure of double toehold pSB1C3 (left) and double trigger pCOLADuetTM-1 plasmid (right).

Figure 2. Electrophoresis of the digested double-toehold pSB1C3 (left) and double-trigger pCOLADuetTM-1 (right).

Figure 3. The sequencing map of double-toehold pSB1C3 (up) and double-trigger pCOLADuetTM-1 (down).

2. Toehold switch function verification.

The double-toehold pSB1C3 and double-trigger pCOLADuetTM-1 plasmids were co-transformed into DH5alpha. The bacteria solution was then cultured 1 hour in 37 ℃ incubator in a double resistance LB plate containing chloramphenicol and kanamycin (Figure4). Positive clone was selected into 6 ml LB. The bacteria solution was shook at 160 rpm in a 37-degree shaker until the OD600 reaches 0.4-0.6, add IPTG, a protein inducer with a final concentration of 1mM, and induce overnight at 160 rpm in a 26-degree shaker. The results showed that the positive control group and double toehold/double trigger group had obvious red fluorescence, these images were captured using a fluorescence microscope (Figure5). Then, 100 l bacteria solution was taken from each group to detect the red fluorescence intensity on Microplate Reader at 587 nm excitation light/610 nm receiving light. The results displayed that double toehold with double trigger group had obvious red fluorescence intensity compared with the Toehold group and the Trigger group (Figure 6). In general, the double toehold switch system can work normally.

Figure 4. Double-toehold pSB1C3 and double-trigger pCOLADuetTM-1 were Individual and co-transferred into DH5α

Figure 5. Observation of bacteria under fluorescence microscope

Figure 6. Statistics of the bacterial fluorescence intensity detection on Microplate Reader.

3. Application

In order to verify methylated specific TFPI2 and SDC2 primers, we use negative genome DNA and simulated colorectal cancer genome DNA as templates, PCR amplification were carried out on real time quantitative PCR instrument with the same number of amplification cycles. Collecting data for statistics showed methylated TFPI2 and SDC2, as the biomarker of colorectal cancer, present higher bar chart in positive samples (Figure 7). These data indicated that the TFPI2 and SDC2 methylation degree has a significant difference between negative and positive samples. The gel electrophoresis of TFPI2 and SDC2 methylation PCR products also showed a significant band in positive sample (Figure 8). Therefore, we infer that TFPI2 and SDC2 can be used as a biomarker for colorectal cancer detection, and there are significant differences in negative and positive samples. In the cell-free protein expression system, we designed the methylated primers of TFPI2 and SDC2, T7 promoter sequence was added to the forward primer and the trigger primer sequence was added to the reverse primer. New primers were synthesized respectively by Tsingke Biology Company. Using negative genome DNA and simulated colorectal cancer patient genome DNA as templates, we performed PCR amplification with the same number of amplification cycles. Then, we transfer the PCR amplified product and the Toehold switch plasmid to the cell-free protein expression system. Incubating the mixture at 37℃ for 3-4 hours before observation. The results showed that positive colorectal cancer samples with TFPI2 and SDC2 methylation specific PCR products group present obvious red fluorescence compare with negative control groups (Figure 9).

Figure 7. Methylated specific TFPI2 and SDC2 primers verified by quantitative PCR.

Figure 8. PCR products were detected by agarose gel electrophoresis; Marker: 100 bp DNA Ladder. Line 1: negative colorectal cancer specimen with ACTB non-methylation specific primers, line 2: positive colorectal cancer samples with ACTB non-methylation specific primers, line 3: positive colorectal cancer samples with TFPI2 methylation specific primers, line 4: positive colorectal cancer samples with SDC2 methylation specific primers.

Figure 9. Functional verification in cell-free protein expression system. Tube1: water; tube2: toehold plasmid only; tube3: trigger plasmid only; tube4: negative colorectal cancer samples with TFPI2 and SDC2 methylation specific PCR product; tube 5: positive colorectal cancer samples with TFPI2 and SDC2 methylation specific PCR product; tube 6: positive control.

4. Summary

In general, we can detect the methylation level of both TFPI2 and SDC2 in patients by observing the intensity of red fluorescence in the cell free system, providing more accurate diagnosis for early screening of CRC compared to last year.