Design

The pET28a plasmid is from our lab's stock, while AsCpf1 is from a biotech company. To construct the plasmid pET-28a-AsCpf1, PCR amplification of the AsCpf1 gene was performed. The pET-28a vector was then subjected to double digestion using BamH I and EcoR I enzymes. The gene fragment was ligated to the linearized vector using a homologous recombination method, followed by transformation into BL21 competent cells. Single colonies were picked from the transformation plate and verified by monoclonal PCR. The amplified products were further sequenced and analyzed by sequence alignment to confirm successful construction of the recombinant plasmid.

To achieve efficient Cas12a protein expression, the recombinant plasmid pET28a-AsCpf1 was transformed into BL21 competent cells. To preserve the plasmids, they were also transformed into DH5α cells. The subsequent AsCpf1 (Cas12a) proteins were extracted, purified, and used for further testing and the construction of the detection platform.

Build

Figure 3. 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 1. The relevant results of pET-28a-AsCpf1 plasmid construction are shown in Figure 3. As depicted in Figure 3A, 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 3B. 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 3C). Transformation efficiency was further verified by monoclonal PCR, as shown in Figure 3D, where the expected band size was observed, confirming the presence of positive clones. Finally, sequence analysis (Figure 3E) revealed that the amplified fragment matched the predicted sequence, thus confirming the successful construction of the pET-28a-AsCpf1 plasmid.

Test

1. Protein Expression

   The protein expression of AsCpf1 (Cas12a) was carried out in the pET-28a-AsCpf1-BL21 strain.

   1. Optimization of IPTG-induced expression at 16 ℃

   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, the protein was purified using a protein purification kit, and both crude and purified protein samples were analyzed by SDS-PAGE.

   

   Figure 4. IPTG-Induced Expression at 16 ℃ with different IPTG concentrations

   The electrophoresis results (Figures 4A and 4B) 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 ℃.

   2. Optimization of IPTG-induced expression at 37 ℃

   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 5. 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 5). 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 ℃.

   3. Induction temperature optimization

   

   Figure 6. IPTG-Induced expression at different temperatures

   Comparing the results of IPTG induction at 16 ℃ (Figure 6A) and 37 ℃ (Figure 6B), 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.

2. Protein functional validation

   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.

   1. Feasibility analysis of the detection platforms

      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.

   2.1.1 Feasibility analysis for fluorescence detection platform

   

   Figure 7. Feasibility analysis for fluorescence detection platform

   As shown in Figure 7, 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.

   2.1.2 Feasibility analysis for test strip detection platform

   

   Figure 8. Feasibility analysis for test strip detection platform

   As shown in Figure 8, 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.

   2. Molecular sensitivity analysis

   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.

   1. RPA product electrophoresis results for molecular sensitivity analysis for c.520G>A and c.6745C>T

   

   Figure 9. RPA product electrophoresis results for molecular sensitivity analysis for c.520G>A and c.6745C>T

   In the electrophoresis gel (Figure 9), 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.

   2. Sensitivity analysis results of the fluorescence detection platform for c.520G>A

   

   Figure 10. 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 10). This indicates that the detection limit of the fluorescence detection system for the c.520G>A site is 10³ copies/µL.

   3. Sensitivity analysis results of the test strip detection platform for c.520G>A

   

   Figure 11. Sensitivity analysis results of the test strip detection platform for c.520G>A

   Observing the test strip results in Figure 11 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.

   4. Sensitivity analysis results of the fluorescence detection platform for c.6745C>T

   

   Figure 12. Sensitivity analysis results of the fluorescence detection platform for c.6745C>T

   The fluorescence detection results for the c.6745C>T site in Figure 12 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.

   5. Sensitivity analysis results of the test strip detection platform for c.6745C>T

   

   Figure 13. 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 13, 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.

   3. Random Sample Analysis

   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).

   1. Random sample analysis for c.520G>A and c.6745C>T (fluorescence detection platform and test strip detection platform)

   

   Figure 14. 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 14. 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.

   2. PCR validation of random samples

   

   Figure 15. PCR validation results of random samples for c.520G>A and c.6745C>T

   Based on the comparison of PCR results (shown in Figure 15) with fluorescence and test strip detection results, the following conclusions can be drawn:

   1. COL7A1 c.520G>A

   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.

   2. COL7A1 c.6745C>T

   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.

Learn

Situation 1:

Problem: During electrophoresis, we observed that two samples were connected.

Analysis: The connection between the two samples is likely due to the well being punctured or scratched during the loading process, causing the samples to merge.

Solution: Carefully check the integrity of the wells before loading the samples. Use a finer pipette tip and ensure gentle and precise loading to avoid puncturing the wells.

Situation 2:

Problem: The plasmid extraction concentration was low.

Analysis: The low plasmid concentration may be due to the low transformation efficiency of the TOP10 cells, which could result in fewer plasmid copies per cell.

Solution: Switch to DH5α competent cells, which generally have higher transformation efficiency and a higher plasmid copy number.

Situation3:

Problem: During the purification process, the wrong reagent was added. The elution buffer was mistakenly used as the wash buffer, causing the sample to be discarded as waste.

Solution: Double-check the reagents before use, ensuring that each reagent is properly labeled and used according to the protocol. Repeat the purification process after correcting the mistake.

Situation 4:

Problem: During the experiment, we observed that after the inoculated bacteria were spread on the culture dish and cultured, the bacterial density was low, and there were scattered scratches on the solid culture dish.

Analysis: The low bacterial density may be due to the insufficient concentration of bacteria applied to the culture dish. The scratches likely resulted from excessive force during the coating process.

Solution: Centrifuge and resuspend the bacterial pellet before coating to remove a portion of the supernatant, thereby increasing the bacterial concentration. When applying the bacteria, slow down and ensure gentle handling to avoid damage to the surface.

Situation 5:

Problem: The bacterial culture growth was too slow.

Analysis: After confirming that the antibiotic was correctly added, the slow growth may be due to a low inoculation volume, poor strain vitality, or the selection of a weak colony.

Solution: Increase the inoculation volume or use a more vigorous strain. We used bacterial cultures from other team members. In the future, we will pay closer attention when selecting colonies to ensure higher viability.

Situation 6:

Problem: Both the marker and the sample were loaded onto the same SDS-PAGE gel.

Solution: Reload the samples, ensuring that the marker and the samples are placed in separate lanes for clear and accurate comparison.

Situation 7:

Problem: During SDS-PAGE electrophoresis, the migration speed was very slow.

Analysis: Upon checking the inner chamber, the buffer level was found to have dropped, which slowed down the electrophoresis process.

Solution: Replenish the electrophoresis buffer to the appropriate level and continue the electrophoresis. Monitor the process closely, adding more buffer as needed to maintain proper electrophoretic conditions.

Situation 8:

Problem: During lateral flow strip analysis, some samples did not show a C line within the first 20 seconds, although a faint C line appeared later.

Analysis: This result is related to the detection principle of Cas12a-based test strips, where strong positive samples contain a high amount of broken ssDNA (forming the T line) and a small amount of intact ssDNA (forming the C line). At the beginning of the assay, there is not enough intact ssDNA accumulated at the C line, which causes the initial absence of the C line.

Solution: Allow more time for the sample to run and accumulate more intact ssDNA for the C line to develop. For very strong positive samples, note that the C line may even disappear, but the T line should be strong. If the T line is weak and no C line is present, the result is invalid.

Design

The pET28a plasmid is from our lab's stock, while gene c.520G (normal sequence) is from a biotech company. To construct the plasmid pET-28a-c.520G, PCR amplification of the c.520G gene fragment was performed. The pET-28a vector was then subjected to double digestion using BamH I and EcoR I enzymes. The amplified gene fragment was ligated into the linearized vector using a homologous recombination method, followed by transformation into DH5α competent cells. Single colonies were picked from the transformation plate and verified by monoclonal PCR. The amplified products were then sequenced and analyzed by sequence alignment to confirm successful construction of the recombinant plasmid.

To obtain a large number of clones, the recombinant plasmid pET-28a-c.520G was transformed into DH5α competent cells, resulting in the acquisition and preservation of the strain pET-28a-c.520G-DH5α.

The construction process is illustrated in Figure 16.

Figure 16. Results of pET-28a-c.520G plasmid construction

Note:

A: Agarose gel electrophoresis results of PCR amplification of the c.520G gene. M: 2 K 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 relevant results of pET-28a-c.520G plasmid construction are shown in Figure 16. As depicted in Figure 16A, 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 16B. 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 16C indicated a high transformation efficiency. Transformation efficiency was further verified by monoclonal PCR, as shown in Figure 16D, where the expected band size was observed, confirming the presence of positive clones. Finally, sequence analysis (Figure 16E) revealed that the amplified fragment matched the predicted sequence, thus confirming the successful construction of the pET-28a-c.520G plasmid.

Test

Plasmid function verification

Figure 17. Electrophoresis results of RPA products using pET-28a-c.520G and pET-28a-c.520G>A as the template

RPA amplification was performed using the pET-28a-c.520G plasmid, followed by the construction and performance evaluation of the subsequent detection platform. For example, in the amplification electrophoresis image shown in Figure 17, 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.

Learn

Situation 1

Problem: After grouping with other teams, it is difficult to distinguish our PCR products.

Analysis: This could be due to a lack of proper labeling, causing sample confusion.

Solution: Ensure clear and visible labeling of each sample before loading them into the gel.

Situation 2

Problem: I observed similar electrophoresis results for our PCR product and another one.

Analysis: The two genes differ by only one nucleotide, so their amplification products are of the same size.

Solution: Understand the difference between these products and confirm they are indeed from different genes. Further analysis can help identify their differences.

Situation 3

Problem: The target band in the RPA electrophoresis gel is hard to separate.

Analysis: RPA products are typically small in size, making it difficult to resolve them properly.

Solution: Use a higher concentration agarose gel for electrophoresis to improve resolution and achieve clearer separation of the target band.

Situation 4

Problem: When using a higher concentration agarose gel, the marker does not spread well.

Analysis: Higher concentration gels create more resistance during electrophoresis, which affects the migration.

Solution: Lower the electrophoresis voltage and increase the running time to achieve better separation.

Design

The pET28a plasmid is from our lab's stock, while gene c.520G>A (mutation sequence) is from a biotech company. For the construction of the pET-28a-c.520 G>A plasmid, PCR amplification of the c .520G>A gene fragment was carried out. The pET-28a vector was then subjected to double digestion using BamH I and EcoR I enzymes to linearize it. The amplified c.520G>A fragment was ligated into the digested vector using a homologous recombination method, followed by transformation into DH5α competent cells. After plating, individual colonies were selected, and monoclonal PCR was performed for initial screening. The cloned products were sequenced and analyzed through sequence alignment to confirm the successful construction of the recombinant plasmid.

To obtain many clones, the recombinant plasmid pET-28a-c.520G>A was transformed into DH5α competent cells, resulting in the acquisition and preservation of the strain pET-28a-c.520G>A-DH5α.

The construction process is illustrated in Figure 18.

Figure 18. The plasmid construction process of pET-28a-c.520G>A

Figure 19. 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.

Construct the plasmid pET-28a-c.520G>A according to Figure 18. The detailed results for the pET-28a-c.520G>A plasmid construction are presented in Figure 19. As shown in Figure 19A, 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 19B, 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 19C, indicating high transformation efficiency. The transformation was further confirmed by monoclonal PCR, shown in Figure 19D, where the expected band size was detected, confirming the presence of positive clones. Lastly, sequence analysis in Figure 19E 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.

Test

Plasmid function verification

RPA amplification was performed using the pET-28a-c.520G>A plasmid, followed by the construction and performance evaluation of the subsequent detection platform. For example, in the amplification electrophoresis image shown in Figure 17, 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.

Learn

Situation 1

Problem: During SDS-PAGE gel preparation, the solution started to solidify before being added to the glass plates.

Analysis: Too much polymerizing agent was added, causing the solution to solidify too quickly.

Solution: Reduce the amount of polymerizing agent to allow more time for the gel to be prepared.

Situation 2

Problem: After adding the lower gel layer in SDS-PAGE preparation, I noticed the liquid level decreased.

Analysis: The gel plates were not properly sealed, leading to leakage.

Solution: Always check for leaks before pouring the gel, ensuring that the plates are tightly sealed.

Situation 3

Problem: After removing the comb in SDS-PAGE preparation, the wells stuck together like bat wings.

Analysis: The comb was not inserted properly, and when removed, the solution had already begun to solidify, causing the wells to merge.

Solution: Insert the comb back quickly after removal before the solution solidifies, or clean the comb and reinsert it to avoid such issues.

Design

The pET28a plasmid is from our lab's stock, while gene c.6745C (normal sequence) is from a biotech company. For the construction of the pET-28a-c.6745C plasmid, a PCR amplification was carried out to obtain the c.6745C gene fragment. The amplified product, which had a size of 310 bp, was inserted into a pET-28a vector that had been digested with BamH I and EcoR I enzymes. This was followed by ligation using a homologous recombination method, and the recombinant plasmid was then introduced into DH5α competent cells. After transformation, individual colonies were picked, and monoclonal PCR was used for verification. The amplified PCR products were sequenced and compared to confirm the successful creation of the recombinant plasmid.

To obtain many clones, the recombinant plasmid pET-28a-c.6745C was transformed into DH5α competent cells, resulting in the acquisition and preservation of the strain pET-28a-c.6745C-DH5α.

The construction process is illustrated in Figure 20.

Figure 20. The construction procedure of the pET-28a-c.6745C plasmid

Figure 21. 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.

Construct the plasmid pET-28a-c.6745C according to Figure 20. The results for the pET-28a-c.6745C plasmid construction are shown in Figure 21. In Figure 21A, 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 21B 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 21C shows the appearance of numerous colonies on the plate, indicating efficient transformation. Monoclonal PCR verification in Figure 21D confirmed the presence of positive clones, as the expected band size was observed. Finally, the sequencing results shown in Figure 21E indicated that the amplified fragment perfectly aligned with the expected sequence, validating the successful construction of the pET-28a-c.6745C plasmid.

Test

Plasmid function verification

Figure 22. Electrophoresis results of RPA products using pET-28a-c.6745C and pET28a-c.6745C>T as the template

RPA amplification was performed using the pET-28a-c.6745C 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.

Learn

Situation 1

Problem: The staining result with Coomassie Brilliant Blue fast stain was not clear.

Analysis: The staining solution had been reused multiple times, which affected its effectiveness.

Solution: We conducted overnight staining and consider using fresh staining solution to improve the staining quality.

Situation 2

Problem: RPA electrophoresis showed target bands as well as additional non-specific bands.

Analysis: RPA products are often longer and more prone to primer-dimer formation or mismatches. Since we cannot distinguish normal and mutated sequences by RPA amplification alone, this is not problematic for our experiment.

Solution: After the RPA reaction, we added the Cas12a cleavage step to specifically identify the target sequence. The non-specific bands will not affect the accuracy of the detection.

Situation 3

Problem: During the test strip experiment, the wrong ssDNA was used for Cas12a cleavage.

Analysis: The ssDNA used for fluorescence detection and for the test strip detection are different. Mixing them up will lead to erroneous results. In a fluorescence detection system, ssDNA must be labeled with fluorescent groups and quenching groups so that a fluorescent signal can be generated in the system after it is cleaved. For test strip detection systems, the ssDNA label must correspond one-to-one with the antibody on the test strip; otherwise, the test strip analysis cannot be performed normally. After using the wrong ssDNA, we repeated the experiment.

Solution: Ensure that the correct ssDNA is used for each detection system. After the mistake, redo the experiment with the proper ssDNA.

Design

The pET28a plasmid is from our lab's stock, while gene c.6745C>T (mutation sequence) is from a biotech company. For the creation of the pET-28a-c.6745C>T plasmid, the PCR amplification was performed using the c.6745C>T gene fragment. The amplified product, which had a size of 310 bp, was then inserted into a pET-28a vector previously digested with BamH I and EcoR I enzymes. The ligation step was completed using a homologous recombination method, and the recombinant plasmid was subsequently introduced into DH5α competent cells. After transformation, individual colonies were selected and monoclonal PCR was conducted to verify the presence of the desired insert. The PCR products were sequenced and aligned to ensure the successful construction of the plasmid.

To obtain many clones, the recombinant plasmid pET-28a-c.6745C>T was transformed into DH5α competent cells, resulting in the acquisition and preservation of the strain pET-28a-c.6745C>T-DH5α.

The construction process is illustrated in Figure 23.

Figure 23. The construction process of the pET-28a-c.6745C>T plasmid

Figure 24. 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 23, with the corresponding results for the pET-28a-c.6745C>T plasmid construction displayed in Figure 24. As seen in Figure 24A, 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 24B, where the expected band sizes were observed, validating successful digestion. After the recombinant plasmid was transformed into DH5α competent cells, Figure 24C demonstrates the appearance of several colonies on the transformation plate, confirming effective transformation. The monoclonal PCR results shown in Figure 24D further supported the presence of positive clones, as indicated by the expected band. Lastly, the sequence alignment results in Figure 24E confirmed that the amplified fragment matched the anticipated sequence, confirming the successful construction of the pET-28a-c.6745C>T plasmid.

Test

Plasmid function verification

RPA amplification was performed using the pET-28a-c.6745C>T 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.

Learn

Situation 11

Problem: Some PCR products from different RPA primers showed weaker electrophoresis bands.

Analysis: The later RPA primers introduced more mismatched sites, which is beneficial for distinguishing normal and mutated sequences in the Cas12a step. However, these mismatches reduce the amplification efficiency.

Solution: The high sensitivity of the Cas12a system compensates for this reduction in amplification efficiency, so continue using the system for detection.

Situation 12

Problem: The detection of the c.520G>A mutation took less time than the c.6745C>T mutation.

Analysis: The RPA primers for the c.6745C>T target introduced more mismatched sites, which reduced the efficiency of crRNA recognition and Cas12a cleavage.

Solution: Increase the Cas12a cleavage time for detecting the c.6745C>T mutation to improve detection accuracy.