In this study, we contributed 10 new parts to iGEM, including 5 basic parts and 5 composite parts. Using these 10 new parts, along with 4 previously existing basic parts, we successfully expressed the AsCpf1 (Cas12a) protein and prepared standard simulated samples for detecting the c.520G>A and c.6745C>T mutations in the COL7A1 gene, which are associated with Dystrophic Epidermolysis Bullosa (DEB). Building on these components, we developed fluorescence detection and test strip detection platforms for the c.520G>A and c.6745C>T mutations, using RPA and engineered Cas12a for expression and purification. These platforms represent a novel, portable, low-cost, and low-equipment-dependent solution, offering a significant contribution to society by improving disease detection rates, enabling early diagnosis, and supporting clinical decision-making, ultimately benefiting human health.
The relevant parts are listed in Table 1. Among them, the first four (BBa_K3521000, BBa_K3521002, BBa_K3521004, BBa_K3584001) are pre-existing parts, while the others are new parts contributed by this study.
Table 1. The relevant parts
|
Part number |
Part name |
Contribution type |
Part type |
|
BBa_K3521000 |
T7 promoter |
basic part |
Regulatory |
|
BBa_K3521002 |
T7 terminator |
basic part |
Terminator |
|
BBa_K3521004 |
pet28a-backbone |
basic part |
Plasmid_Ba |
|
BBa_K3584001 |
His tag-Lac operate-T7 tag |
basic part |
Tag |
|
BBa_25GBCNYZ |
AsCpf1 |
basic part |
Coding |
|
BBa_250ZZSEH |
pET28a-AsCpf1 |
composite part |
Plasmid |
|
BBa_259OXPJX |
c.520G>A |
basic part |
Coding |
|
BBa_25QUOU62 |
c.520G |
basic part |
Coding |
|
BBa_2593FMVL |
c.6745C>T |
basic part |
Coding |
|
BBa_2584X1ZC |
c.6745C |
basic part |
Coding |
|
BBa_25A2GFYI |
pET28a-c.520G>A |
composite part |
Plasmid |
|
BBa_25XMA9IE |
pET28a-c.520G |
composite part |
Plasmid |
|
BBa_25LN1XT0 |
pET28a-c.6745C>T |
composite part |
Plasmid |
|
BBa_25WJ6OVG |
pET28a-c.6745C |
composite part |
Plasmid |
Name: AsCpf1
Part number: BBa_25GBCNYZ
Base Pair: 3936 bp
Property: coding
Usage and Biology:
AsCpf1, also known as Cas12a, is an enzyme belonging to CRISPR system from Class 2, Type V-A. It works with a single RNA guide and is widely used in gene editing by recognizing specific sequences on DNA. Unlike Cas9, AsCpf1 needs only one crRNA to find and cut DNA, so it does not require another tracrRNA. This makes designing guide RNAs easier and simpler. It recognizes PAM sequences with TTN, which allows it to target parts of the genome well and generates staggered double-strand breaks with 5' overhangs, leading to precise DNA insertions or deletions. As the experiment requires to cut double-stranded DNA, Cas14 cannot be used as it cuts single-stranded DNA. Another advantage of AsCpf1 is trans-cleavage activity, which can help AsCpf1 use one probe to detect the mutated gene locus. Zhang and colleagues (2021) developed a Cas12a system by constructing a Cas12a plasmid and pre-crRNA array plasmid. They demonstrated that one crRNA can edit two genetic loci and used Cas12a to process. This can simplfy construction steps and make efficiently.
Experimental approach:
Figure 1. Amplification results of the AsCpf1 gene
Note:
A: Gene amplification process of AsCpf1.
B: The gel electrophoresis results of the PCR products for AsCpf1. M: 5 K DNA Marker, N: negative control.
C: Gene sequence map of AsCpf1.
D: Sequence alignment results.
The gene amplification of AsCpf1 was successfully carried out, as designed in Figure 1A. The gel electrophoresis results of the PCR products are displayed in Figure 1B. A single bright band corresponding to the amplified AsCpf1 gene was observed at 3936 bp, as expected. The negative control (N) showed no amplification, confirming the specificity of the PCR.
To validate the amplified fragment, we compared the sequence obtained from the PCR product with the gene sequence map in Figure 1C. The sequence alignment, shown in Figure 1D, confirmed that the amplified fragment was identical to the expected AsCpf1 sequence, thereby verifying the accuracy of the amplification. We successfully obtained the full-length AsCpf1 gene
Reference:
Mendoza, C. S., Findlay, A., & Judelson, H. S. (2023). A Variant of LbCas12a and Elevated Incubation Temperatures Enhance the Rate of Gene Editing in the Oomycete Phytophthora infestans. Molecular Plant-Microbe Interactions, 36(11), 677–681. https://doi.org/10.1094/mpmi-05-23-0072-sc
Zhang, X., Gu, S., Zheng, X., Peng, S., Li, Y., Lin, Y., & Liang, S. (2021). A Novel and Efficient Genome Editing Tool Assisted by CRISPR-Cas12a/Cpf1 for Pichia pastoris. ACS Synthetic Biology, 10(11), 2927–2937. https://doi.org/10.1021/acssynbio.1c00172
Name: c.520G
Part number: BBa_25QUOU62
Base Pair: 362 bp
Property: Coding
Origin: homo sapiens
Usage and Biology:
It functions as an anchoring fibril between the external epithelia and the underlying stroma.
COL7A1 c.520G represents the wild allele encoding type VII collagen, a major component of anchoring fibrils at the dermal and epidermal junction. Biologically, the presence of this allele ensures the correct folding of collagen VII and contributes to stable skin adhesion. In research usage, c.520G is frequently used as a control reference when comparing pathogenic variants in patient samples. It provides the baseline sequence for mutation detection assays and serves as a template in studies of CRISPR-based correction strategies targeting COL7A1.
Experimental Approach:
Figure 2. Amplification results of the C.520G gene
Note:
A: Gene amplification process of C.520G.
B: The gel electrophoresis results of the PCR products for C.520G. M: 5 K DNA Marker, N: negative control.
C: Gene sequence map of C.520G.
D: Sequence alignment results.
To amplify the c.520G gene, we designed the gene amplification process for c.520G as shown in Figure 2A. The gene sequence of c.520G was obtained and is displayed in Figure 2C. Next, we added the primers PCR-c.520G-F and PCR-c.520G-R, along with other necessary components, to amplify c.520G by PCR. The amplified gene fragment was observed using agarose gel electrophoresis, where we observed a single bright band over 250 bp, corresponding to the expected 362 base pairs, as shown in Figure 2B.
To confirm that the amplified fragment was our target, we had the sample sequenced by a service provider. The sequencing result is shown in Figure 2D. Comparing Figure 2D with Figure 2C, we found that the sequences were identical, confirming the successful amplification of the correct gene fragment.
Referennce:
Pironon, N., Bourrat, E., Prost, C., Chen, M., Woodley, D. T., Titeux, M., & Hovnanian, A. (2024). Splice modulation strategy applied to deep intronic variants in COL7A1 causing recessive dystrophic epidermolysis bullosa. Proceedings of the National Academy of Sciences, 121(35). https://doi.org/10.1073/pnas.2401781121
Name: c.520G>A
Part number: BBa_259OXPJX
Base Pair: 362 bp
Property: Coding
Origin: Homo sapiens
Usage and Biology:
COL7A1 c.520G>A is a point mutation that introduces an amino acid substitution in the N-terminal region of collagen VII. Biologically, this change may disrupt protein folding and compromise anchoring fibril formation, leading to fragile skin and blistering typical of dystrophic epidermolysis bullosa. From a usage perspective, c.520G>A is often studied as a pathogenic variant in genetic diagnosis, functional assays, and therapeutic evaluation. It provides a defined target site for CRISPR-mediated correction and has been applied in the design of specific probes for molecular diagnostics.
Experimental Approach:
Figure 3. Amplification results of the C.520G>A gene
Note:
A: Gene amplification process of C.520G>A.
B: The gel electrophoresis results of the PCR products for C.520G>A. M: 5 K DNA Marker, N: negative control.
C: Gene sequence map of C.520G>A.
D: Sequence alignment results.
To amplify the c.520G>A gene, we designed the gene amplification process for c.520G>A, as shown in Figure 3A. The gene sequence of c.520G>A was obtained and is displayed in Figure 3C. Next, we added the primers PCR-c.520G>A-F and PCR-c.520G>A-R, along with other necessary components, to amplify c.520G>A by PCR. The amplified gene fragment was observed using agarose gel electrophoresis, where a single bright band over 250 bp was detected, corresponding to the expected 362 base pairs, as shown in Figure 3B.
To confirm that the amplified fragment was our target, we had the sample sequenced by a service provider. The sequencing result is shown in Figure 3D. By comparing Figure 3D with Figure 3C, we found that the sequences were identical, confirming that the correct gene fragment had been successfully amplified.
Reference:
Kocher, T., March, O. P., Bischof, J., Liemberger, B., Hainzl, S., Klausegger, A., Hoog, A., Strunk, D., Bauer, J. W., & Koller, U. (2020). Predictable CRISPR/CAS9-Mediated COL7A1 reframing for dystrophic epidermolysis Bullosa. Journal of Investigative Dermatology, 140(10), 1985-1993.e5. https://doi.org/10.1016/j.jid.2020.02.012
Name: c.6745C
Part number: BBa_2584X1ZC
Base Pair: 310 bp
Property: Coding
Origin: homo sapiens
Usage and Biology:
The COL7A1 c.6745C allele represents the wild-type sequence within the triple-helical domain of type VII collagen. This domain is essential for assembling stable anchoring fibrils, which support dermal–epidermal cohesion and preserve skin integrity under mechanical stress. In research and diagnostic contexts, c.6745C is typically used as a reference sequence. It provides a baseline for comparison with the pathogenic c.6745C>T variant in studies of genotype–phenotype relationships and is also employed to confirm the reliability of sequencing assays and gene-editing approaches.
Experimental appraoch:
Figure
4. Amplification results of the C.6745C gene
Note:
A: Gene amplification process of C.6745C.
B: The gel electrophoresis results of the PCR products for C.6745C. M: 5 K DNA Marker, N: negative control.
C: Gene sequence map of C.6745C.
D: Sequence alignment results.
To amplify the c.6745C gene, we designed the gene amplification process for c.6745C, as shown in Figure 4A, and obtained the corresponding gene sequence and its map, displayed in Figure 4C. Next, we added the primers PCR-c.6745C-F and PCR-c.6745C-R, along with other necessary components, to amplify c.6745C by PCR. The amplified gene fragment was analyzed using agarose gel electrophoresis, where a single bright band over 250 bp, corresponding to the expected 310 base pairs, was observed, as shown in Figure 4B.
To confirm that the amplified fragment was the correct target, we outsourced sequencing of the sample. The sequencing results are displayed in Figure 4D. By comparing Figure 4D with Figure 4C, we confirmed that the sequences were identical, confirming the correct amplification of the gene fragment.
Reference:
Bruckner-Tuderman, L., Höpfner, B., & Hammami-Hauasli, N. (1999). Biology of anchoring fibrils: lessons from dystrophic epidermolysis bullosa. Matrix Biology, 18(1), 43–54. https://doi.org/10.1016/s0945-053x(98)00007-9
Name: c.6745C>T
Part number: BBa_2593FMVL
Base Pair: 310 bp
Property: Coding
Origin: homo sapiens
Usage and Biology:
The COL7A1 c.6745C>T variant causes a missense change in the triple-helical domain of type VII collagen, a region essential for maintaining its structural integrity. This mutation disrupts the normal assembly of anchoring fibrils and is commonly linked to recessive dystrophic epidermolysis bullosa, leading to fragile skin and persistent wounds. In research and clinical practice, c.6745C>T is considered a clearly defined pathogenic mutation and is frequently used in diagnostic assays, protein functional studies, and the development of therapeutic strategies. Its precise nucleotide change also makes it a useful model for testing gene-editing approaches and designing assays for accurate mutation detection and potential correction.
Experimental Approach:
Figure 5. Amplification results of the C.6745C>T gene
Note:
A: Gene amplification process of C.6745C>T.
B: The gel electrophoresis results of the PCR products for C.6745C>T. M: 5 K DNA Marker, N: negative control.
C: Gene sequence map of C.6745C>T.
D: Sequence alignment results.
To amplify the c.6745C>T gene, we designed the gene amplification process for c.6745C>T, as shown in Figure 5A, and obtained the corresponding gene sequence map, displayed in Figure 5C. Next, we added the primers PCR-c.6745C>T-F and PCR-c.6745C>T-R, along with other necessary components, to amplify c.6745C>T by PCR. The amplified gene fragment was analyzed using agarose gel electrophoresis, where a single bright band over 250 bp, corresponding to the expected 310 base pairs, was observed, as shown in Figure 5B.
To confirm that the amplified fragment was the correct target, we outsourced sequencing of the sample. The sequencing results are displayed in Figure 5D. By comparing Figure 5D with Figure 5C, we confirmed that the sequences were identical, ensuring the correct amplification of the gene fragment.
Reference:
Dang, N., & Murrell, D. F. (2008). Mutation analysis and characterization of COL7A1 mutations in dystrophic epidermolysis bullosa. Experimental Dermatology, 17(7), 553–568. https://doi.org/10.1111/j.1600-0625.2008.00723.x
Name: pET28a-AsCpf1 BBa_250ZZSEH
Base Pair: 9289 bp
Property: plasmid
Figure 6. The plasmid construction process of pET-28a-AsCpf1
Figure 7. Results of pET-28a-AsCpf1 plasmid construction
Note:
A: Agarose gel electrophoresis results of PCR amplification of the AsCpf1 gene. M: 5K DNA Marker, N: negative control.
B: Double digestion results of pET-28a using BamH I and EcoR I.
C: Culture plate for transforming competent cells with recombinant plasmids.
D: Monoclonal PCR verification of transformation results.
E. Sequence alignment results of recombinant plasmid pET-28a-AsCpf1.
The construction process is illustrated in Figure 6. The relevant results of pET-28a-AsCpf1 plasmid construction are shown in Figure 7. As depicted in Figure 7A, the PCR amplification of the AsCpf1 gene produced a single band around 4000 bp, which aligns with the expected size of 3936 bp, confirming successful amplification of the AsCpf1 gene. The double digestion results of the pET-28a vector are presented in Figure 7B. Upon examination of the digestion products, the presence of the expected band size and comparison with other lanes verified successful digestion. Subsequently, the recombinant plasmid was transformed into BL21 competent cells, and the appearance of numerous single colonies on the transformation plate indicated a high transformation efficiency (Figure 7C). Transformation efficiency was further verified by monoclonal PCR, as shown in Figure 7D, where the expected band size was observed, confirming the presence of positive clones. Finally, sequence analysis (Figure 7E) revealed that the amplified fragment matched the predicted sequence, thus confirming the successful construction of the pET-28a-AsCpf1 plasmid.
The protein expression of AsCpf1 (Cas12a) was carried out in the pET-28a-AsCpf1-BL21 strain.
To optimize the IPTG-induced expression of AsCpf1, pET-28a-AsCpf1-BL21 acterial cultures were grown to an OD600 of 0.6, followed by IPTG induction at concentrations of 0 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, and 2 mM. The cultures were then incubated at 16 ℃ with shaking for 20 hours. After induction, protein was purified using a protein purification kit, and both crude and purified protein samples were analyzed by SDS-PAGE.
Figure 8. IPTG-Induced Expression at 16 ℃ with different IPTG concentrations
The electrophoresis results (Figures 8A and 8B) showed clear overexpression of the target protein in the crude sample, with increasing IPTG concentrations leading to stronger protein bands. After purification, the impurities in the crude protein were almost entirely removed, leaving predominantly the target protein. The intensity of the bands in both the crude and purified samples followed a consistent trend with the increasing IPTG concentration. The sample induced with 0.8 mM IPTG exhibited the strongest band, both in the crude and purified forms, indicating that 0.8 mM IPTG is the optimal concentration for protein expression at 16 ℃.
For higher-temperature expression, cultures were induced with IPTG at concentrations of 0 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, and 2 mM, after reaching an OD600 of 0.6. The cultures were incubated at 37 ℃ with shaking for 3 hours. Afterward, protein was purified using the same protocol as the 16 ℃ induction, and the samples were analyzed by SDS-PAGE.
Figure 9. IPTG-Induced Expression at 37 ℃ with different IPTG concentrations
The electrophoresis gel for the 37 ℃ induction revealed weaker overall band intensities compared to the 16 ℃ induction (Figure 9). As the IPTG concentration increased, the band intensity did not show significant changes. However, the samples induced with 0.5 mM and 2 mM IPTG showed relatively stronger bands, suggesting these concentrations are optimal for expression at 37 ℃. Although purification reduced some of the impurities, the overall protein expression at 37 ℃ was obviously weaker than at 16 ℃.
Figure 10. IPTG-Induced Expression at different temperatures
Comparing the results of IPTG induction at 16 ℃ (Figure 10A) and 37 ℃ (Figure 10B), it was observed that protein expression was significantly higher at the lower temperature (16 ℃), especially at 0.8 mM IPTG. The overall band intensity at 16 ℃ was relatively high, and the purification process removed most impurities, leaving a clear target protein band. In contrast, protein expression at 37 ℃ was weaker, even with higher IPTG concentrations, indicating that the lower temperature (16 ℃) provides better conditions for AsCpf1 expression.
The AsCpf1 (Cas12a) protein, obtained through synthetic biology methods, was purified and used in combination with RPA for the construction of detection platforms targeting COL1A1 gene mutations c.520G>A and c.6745C>T for the diagnosis of dermatosparaxis Ehlers-Danlos syndrome (DEB). The detection process involves fetal DNA acquisition, RPA amplification, and Cas12a cleavage. Using different ssDNA reporters, we constructed both a fluorescence detection platform and a test strip detection platform.
In our experiments, RPA primers were first screened, followed by optimization of the reaction time for Cas12a cleavage of RPA products. Ultimately, we finalized the components of the reaction system and established the detection platforms. During this process, the Cas12a protein, obtained through synthetic biology methods, demonstrated excellent cleavage activity. Part of the experimental results, which validate the Cas12a activity and the effectiveness of the detection platform we developed, are shown below.
For the two mutation sites c.520G>A and c.6745C>T, RPA amplification, Cas12a cleavage reaction, fluorescence detection, and test strip analysis were performed on normal and mutant sequences under optimized conditions.
- Feasibility analysis for fluorescence detection platform
- Feasibility analysis for test strip detection platform
Figure 11. Feasibility analysis for fluorescence detection platform
As shown in Figure 11, for the c.520G>A and c.6745C>T mutation sites, the fluorescence signals from the mutant sequences were significantly higher than those from the normal sequences, indicating that the optimized fluorescence detection system can effectively distinguish between normal and mutant sequences. This detection system is feasible.
Figure 12. Feasibility analysis for test strip detection platform
As shown in Figure 12, for the c.520G>A and c.6745C>T mutation sites, the test strips for the mutant sequences display both a control line (C line) and a distinct test line (T line), while the normal sequences and blank controls only show the control line (C line). This indicates that the optimized test strip detection system can effectively distinguish between normal and mutant sequences, demonstrating the feasibility of this detection system.
Conclusion:
Both the fluorescence and test strip detection platforms effectively distinguish between normal and mutant sequences at the c.520G>A and c.6745C>T mutation sites. The optimized systems demonstrated clear differentiation, confirming their feasibility for mutation detection.
For the two mutation sites c.520G>A and c.6745C>T, to investigate the analytical sensitivity of the detection system, plasmid samples containing different copy numbers of the mutated sequences were subjected to RPA amplification, Cas12a cleavage reaction, fluorescence detection, and test strip analysis under optimized conditions.
The plasmid concentrations used were: 10⁸ copies/μL, 10⁵ copies/μL, 10⁴ copies/μL, 10³ copies/μL, 10² copies/μL, 10¹ copies/μL, and 10⁰ copies/μL.
- RPA product electrophoresis results for molecular sensitivity analysis for c.520G>A and c.6745C>T
- Sensitivity analysis results of the fluorescence detection platform for c.520G>A
- Sensitivity analysis results of the test strip detection platform for c.520G>A
- Sensitivity analysis results of the fluorescence detection platform for c.6745C>T
- Sensitivity analysis results of the test strip detection platform for c.6745C>T
Figure 13. RPA product electrophoresis results for molecular sensitivity analysis for c.520G>A and c.6745C>T
In the electrophoresis gel (Figure 13), the RPA amplification products of samples 108, 106, and 105 copies/µL showed bands that were consistent with the expected size. As the plasmid concentration decreased, the electrophoretic bands of the RPA products gradually weakened until they disappeared. This is due to the limited analytical capacity of RPA amplification combined with agarose gel electrophoresis. In summary, the appearance of the target band in the high-concentration samples and the absence of bands in the blank control group indicate that the RPA amplification reaction proceeded normally.
Figure 14. Sensitivity analysis results of the fluorescence detection platform for c.520G>A
Analysis of the fluorescence detection results for the c.520G>A site revealed that as the plasmid concentration decreased, the fluorescence signal showed an overall decreasing trend. The fluorescence value of the 103 copies/µL sample showed a significant difference compared to the negative control, whereas the 102 copies/µL sample did not (Figure 14). This indicates that the detection limit of the fluorescence detection system for the c.520G>A site is 103 copies/µL.
Figure 15. Sensitivity analysis results of the test strip detection platform for c.520G>A
Observing the test strip results in Figure 15 for the c.520G>A site, it was found that as the plasmid concentration decreased, the strength of the T line on the test strip showed an overall decreasing trend. The 103 copies/µL sample displayed a clear T line, while the 102 copies/µL sample did not. This indicates that the detection limit of the test strip system for the c.520G>A site is 103 copies/µL.
Figure 16. Sensitivity analysis results of the fluorescence detection platform for c.6745C>T
The fluorescence detection results for the c.6745C>T site in Figure 16 showed that as the plasmid concentration decreased, the fluorescence signal overall exhibited a decreasing trend. The fluorescence value of the 105 copies/µL sample showed a significant difference from the negative control, while the 104 copies/µL sample displayed some fluorescence, but the data analysis revealed no significant difference compared to the negative control. This suggests that the detection limit of the fluorescence detection system for the c.6745C>T site is 105 copies/µL.
Figure 17. Sensitivity analysis results of the test strip detection platform for c.6745C>T
Observing the test strip results for the c.6745C>T site, it was found that as the plasmid concentration decreased, the strength of the T line on the test strip also showed a decreasing trend. As shown in Figure 17, the 104 copies/µL sample displayed a clear T line, while the 103 copies/µL sample did not. This indicates that the detection limit of the test strip system for the c.6745C>T site is 104 copies/µL.
Conclusion:
The molecular sensitivity analysis reveals that the fluorescence detection system for the c.520G>A site has a detection limit of 10³ copies/µL, while the test strip system has the same limit. For the c.6745C>T site, the fluorescence detection system has a limit of 10⁵ copies/µL, and the test strip system has a limit of 10⁴ copies/µL.
For the two target sites, c.520G>A and c.6745C>T, standard plasmids containing both mutated and non-mutated sequences at varying concentrations (≥104 copies/µL) were randomly mixed. A total of 12 random samples were prepared, and the constructed method was used for analysis. All random samples were subjected to RPA amplification, Cas12a cleavage reaction, fluorescence detection, and test strip analysis under optimized conditions. Positive and negative controls were also set up in the experiment. PCR amplification is used to verify the presence of relevant genes (it cannot distinguish whether they are mutant sequences).
- Random sample analysis for c.520G>A and c.6745C>T (fluorescence detection platform and test strip detection platform)
- PCR validation of random samples
Figure 18. Random sample analysis results for c.520G>A and c.6745C>T
The results of the random sample analysis for the c.520G>A and c.6745C>T targets are displayed in Figure 18. Both the fluorescence detection and test strip detection methods yielded consistent results, effectively distinguishing the presence or absence of mutated sequences. This indicates that the detection system can analyze actual samples.
Figure 19. PCR validation results of random samples for c.520G>A and c.6745C>T
Based on the comparison of PCR results (shown in Figure 19) with fluorescence and test strip detection results, the following conclusions can be drawn:
- COL7A1 c.520G>A
- COL7A1 c.6745C>T
Samples 2, 3, 5, 7, 8, 10, 11, and 12 contain the COL7A1 c.520 near gene segment. Among these, samples 2, 3, 7, 10, and 11 contain the COL7A1 c.520G>A (mutated sequence), while samples 5, 8, and 12 contain the COL7A1 c.520G (non-mutated sequence). Samples 1, 4, 6, and 9 do not contain the COL7A1 c.520 near gene segment.
Samples 2, 3, 5, 7, 10, 11, and 12 contain the COL7A1 c.6745C near gene segment. Among these, samples 2, 3, 7, 10, and 11 contain the COL7A1 c.6745C>T (mutated sequence), while samples 5 and 12 contain the COL7A1 c.6745C (non-mutated sequence). Samples 1, 4, 6, 8, and 9 do not contain the COL7A1 c.6745C near gene segment.
These results confirm that the detection system can accurately identify both the presence of the targeted gene segments and the mutations associated with COL7A1.
Name: pET28a-c.520G
Part number: BBa_25XMA9IE
Base Pair: 5715 bp
Property: plasmid
Figure 20. The plasmid construction process of pET-28a-c.520G
Figure 21. Results of pET-28a-c.520G plasmid construction
Note:
A: Agarose gel electrophoresis results of PCR amplification of the c.520G gene. M: 2K DNA Marker, N: negative control.
B: Double digestion results of pET-28a using BamH I and EcoR I.
C: Culture plate for transforming competent cells with recombinant plasmids.
D: Monoclonal PCR verification of transformation results.
E: Sequence alignment results of recombinant plasmid pET-28a-c.520G.
The construction process is illustrated in Figure 20. The relevant results of pET-28a-c.520G plasmid construction are shown in Figure 21. As depicted in Figure 21A, the PCR amplification of the c.520G gene produced a single band around 362 bp, which aligns with the expected size of 362 bp, confirming successful amplification of the c.520G gene. The double digestion results of the pET-28a vector are presented in Figure 21B. Upon examination of the digestion products, the presence of the expected band size and comparison with other lanes verified successful digestion. Subsequently, the recombinant plasmid was transformed into DH5α competent cells, and the appearance of numerous single colonies on the transformation plate in Figure 21C indicated a high transformation efficiency. Transformation efficiency was further verified by monoclonal PCR, as shown in Figure 21D, where the expected band size was observed, confirming the presence of positive clones. Finally, sequence analysis (Figure 21E) revealed that the amplified fragment matched the predicted sequence, thus confirming the successful construction of the pET-28a-c.520G plasmid.
Figure 22. Electrophoresis results of RPA products using pET-28a-c.520G and pET-28a-c.520G>A as the template
RPA amplification was performed using this plasmid, followed by the construction and performance evaluation of the subsequent detection platform. For example, in the amplification electrophoresis image shown in Figure 22, the expected-sized amplification products were observed. Combined with sequencing results, this indicates that the plasmid can be used as a standard reference sample for the DEB detection platform.
Name: pET28a-c.520G>A
Part name: BBa_25A2GFYI
Base Pair: 5715 bp
Property: plasmid
Figure 23. The plasmid construction process of pET-28a-c.520G>A
Figure 24. Results of pET-28a-c.520G>A plasmid construction
Note:
A: Agarose gel electrophoresis showing the PCR amplification of the c.520G>A gene. M: 2K DNA Marker, N: negative control.
B: Double digestion results of pET-28a vector using BamH I and EcoR I.
C: Transformation plate showing DH5α competent cells containing recombinant plasmids.
D: Monoclonal PCR results for transformation verification.
E: Sequence alignment results confirming the recombinant plasmid.
The process of plasmid construction is outlined in Figure 23. The detailed results for the pET-28a-c.520G>A plasmid construction are presented in Figure 24. As shown in Figure 24A, the PCR amplification of the c.520G>A gene generated a single band around 362 bp, which corresponds with the expected fragment size, confirming successful gene amplification. The results of the double digestion of the pET-28a vector are shown in Figure 24B, where the digestion products reveal the presence of the expected fragment sizes, confirming proper digestion. The transformation of the recombinant plasmid into DH5α cells resulted in the formation of numerous single colonies, as illustrated in Figure 24C, indicating high transformation efficiency. The transformation was further confirmed by monoclonal PCR, shown in Figure 24D, where the expected band size was detected, confirming the presence of positive clones. Lastly, sequence analysis in Figure 24E confirmed that the sequence of the amplified c.520G>A fragment matched the expected sequence, validating the successful construction of the pET-28a-c.520G>A plasmid.
RPA amplification was performed using this plasmid, followed by the construction and performance evaluation of the subsequent detection platform. For example, in the amplification electrophoresis image shown in Figure 22, the expected-sized amplification products were observed. Combined with sequencing results, this indicates that the plasmid can be used as a standard reference sample for the DEB detection platform.
Name: pET28a-c.6745C
Part name: BBa_25WJ6OVG
Base Pair: 5663 bp
Property: plasmid
Figure 25. The construction procedure of the pET-28a-c.6745C plasmid
Figure 26. Results of pET-28a-c.6745C plasmid construction
Note:
A: PCR amplification results of the c.6745C gene. M: 2K DNA Marker, N: negative control.
B: Double digestion of pET-28a vector with BamH I and EcoR I enzymes.
C: Colony formation on the plate after transformation with recombinant plasmids.
D: Monoclonal PCR verification of the transformation results.
E: Sequence alignment of the recombinant plasmid pET-28a-c.6745C.
The plasmid construction process is illustrated in Figure 25. The results for the pET-28a-c.6745C plasmid construction are shown in Figure 26. In Figure 26A, PCR amplification of the c.6745C gene produced a single band around 310 bp, which matched the expected size, confirming the successful amplification of the c.6745C gene. Figure 26B shows the results of the double digestion of the pET-28a vector. The digestion products revealed the expected band sizes, confirming the success of the digestion process. Following this, the recombinant plasmid was transformed into DH5α competent cells, and Figure 26C shows the appearance of numerous colonies on the plate, indicating efficient transformation. Monoclonal PCR verification in Figure 26D confirmed the presence of positive clones, as the expected band size was observed. Finally, the sequencing results shown in Figure 26E indicated that the amplified fragment perfectly aligned with the expected sequence, validating the successful construction of the pET-28a-c.6745C plasmid.
Figure 27. Electrophoresis results of RPA products using pET-28a-c.6745C and pET28a-c.6745C>T as the template
RPA amplification was performed using this plasmid, followed by the construction and performance evaluation of the subsequent detection platform. For example, in the amplification electrophoresis image shown in Figure 27, the expected-sized amplification products were observed. Combined with sequencing results, this indicates that the plasmid can be used as a standard reference sample for the DEB detection platform.
Name: pET28a-c.6745C>T
Part name: BBa_25LN1XT0
Base Pair: 5663 bp
Property: plasmid
Figure 28. The construction process of the pET-28a-c.6745C>T plasmid
Figure 29. Results of the pET-28a-c.6745C>T plasmid construction
Note:
A: PCR amplification of the c.6745C>T gene. M: 2K DNA Marker, N: negative control.
B: Double digestion of pET-28a vector with BamH I and EcoR I.
C: Colony formation on the transformation plate after recombinant plasmid introduction.
D: Monoclonal PCR verification of the transformation.
E: Sequence alignment of the recombinant plasmid pET-28a-c.6745C>T.
The plasmid construction process is shown in Figure 28, with the corresponding results for the pET-28a-c.6745C>T plasmid construction displayed in Figure 29. As seen in Figure 29A, PCR amplification of the c.6745C>T gene generated a distinct band at 310 bp, confirming the successful amplification of the gene. The results of the double digestion of the pET-28a vector are presented in Figure 29B, where the expected band sizes were observed, validating successful digestion. After the recombinant plasmid was transformed into DH5α competent cells, Figure 29C demonstrates the appearance of several colonies on the transformation plate, confirming effective transformation. The monoclonal PCR results shown in Figure 29D further supported the presence of positive clones, as indicated by the expected band. Lastly, the sequence alignment results in Figure 29E confirmed that the amplified fragment matched the anticipated sequence, confirming the successful construction of the pET-28a-c.6745C>T plasmid.
RPA amplification was performed using this plasmid, followed by the construction and performance evaluation of the subsequent detection platform. For example, in the amplification electrophoresis image shown in Figure 27, the expected-sized amplification products were observed. Combined with sequencing results, this indicates that the plasmid can be used as a standard reference sample for the DEB detection platform.
Enrich the sample source
- Replace blood sampling with urine as the sample source. Because Urine is rich in cell-free DNA (cfDNA) and is a promising non-invasive alternative. So, it can improve patient compliance, reduces sampling risks(Non-invasive), and enables future convenient testing.
- Expansion of Detection Targets (Multiplexing):Move beyond detecting monogenic mutations for Dystrophic Epidermolysis Bullosa (DEB). We can incorporate additional crRNAs into the CRISPR cleavage system. So, it enable simultaneous detection of key mutations associated with the three other major EB subtypes like JEB, KEB, EBS(polygenic mutation). Maybe it has capability for other genetic diseases with similar genetic backgrounds.
- Improve the accuracy of our experimental resulus: Replace test strip markers with fluorescent nanobeads (100× sensitivity gain; requires UV/instrument readout).
Use dUTP in amplification + pre-experiment UDG treatment to degrade contaminants. Physically separate processing, amplification, and detection zones (sterile preferred) to prevent aerosol contamination. Integrate qPCR for CRISPR assistance to quantitatively verify sample DNA quality. Establish optimal diagnostics via ROC curve analysis (TPR vs FPR) to calculate AUC and determine best cut-off value.
Through the aforementioned methods (utilizing non-invasive sampling and multiple mutation sites to enhance experimental accuracy), we believe the next phase will achieve: A revolutionary shift from blood to urine samples, fully realizing non-invasive sampling to reduce psychological burdens on pregnant women. Detection of all four major EB subtypes across the platform by incorporating additional crRNA designs. Significant enhancement of test results via the integrated CRISPR-qPCR fusion system, dUTP/UDG anti-contamination protocols, laboratory zoning, ROC curve optimization, and fluorescent nanobeads. This project will accelerate the historic transition in EB diagnosis—from single-gene invasive testing to a non-invasive urine screening platform with multiplexed EB subtype diagnosis—establishing a new global benchmark for precision genetic disease management. Upholding iGEM’s mission of ‘Synthetic Biology for a Better World’ we will transform this concept into a deployable platform.
