In this project, we are committed to providing consumers with a rapid and reliable solution to quickly detect the presence of Staphylococcus aureus and Pseudomonas aeruginosa in their daily lives. This application extends beyond cosmetics. Leveraging the state-of-the-art CRISPR/Cas12a gene editing technology, we have cost-effectively transcribed Cas12 protein in vitro and synthesized sgRNA to guide Cas12 protein cleavage. Combined with gene fluorescence probes, we have successfully achieved rapid detection of specific pathogenic gene segments on simulated Escherichia coli, serving as a tool to identify Staphylococcus aureus. The fluorescence intensity observed by the naked eye can determine negativity, positivity, and approximate bacterial quantities. This system is user-friendly and easy to use.
Protein Cas12a expression
First of all, we need Cas12a protein and plasmid pET28a-FnCas12a (BBa_K4304007, provided by iGEM22_YkPaO, SubCat Academy) was obtained from the company laboratories. We transformed the pET28a-FnCas12a plasmid into E. coli BL21(DE3) and cultured overnight. Then we transferred the medium into 300mL fresh LB culture medium. When the OD600 was around 0.6, we added IPTG to induce FnCas12a expression at 16℃ for 12 hours. Then FnCas12a was extracted and purified by His-tag. The size of protein FnCas12a is about 130kDa. The result of SDS-PAGE showed that the size of the FnCas12a protein is between 100kDa and 160kDa. So the protein FnCas12a was successfully expressed.
Figure 1. SDS-PAGE analysis of FnCas12a protein
Construct design and build 1
pUC57-femA: BBa_K4926002
We designed a plasmid containing femA. The DNA fragment is from the genome of Staphylococcus aureus. In order to construct plasmid, we let the company synthesize the DNA fragment femA, which was inserted into pUC57 plasmid.
We transformed the pUC57-femA into the E.coli DH5α. We obtained the E.coli DH5α with target fragments of femA (Fig 2)., Then we performed TAE agarose gel electrophoresis to verify that the plasmid presented in the E.coli DH5α. As shown in Figure 3, we got lanes which length were same with femA.
Figure 2. pUC57-femA in DH5α
Figure 3. Bacteria PCR of femA.
Function test 1
Figure 4. Graph of efficiency comparison of sgRNAs
We used T7 in vitro transcription kit to obtain three different sgRNA of femA. In order to verify whether Cas12a could recognize and cleave the DNA fragments, we mixed Cas12a, sgRNA of femA and the plasmids. We tested the fluorescence of the sgRNA after incubation. In the graph, the vertical axis represents the relative intensity, which means the change in the intensity of the fluorescence. Faster decreasing slope means higher efficiency in uncoiling the plasmid. Since all the sgRNA shows decreasing slope, all of them successfully react with the plasmids, which means our experiment succeed. The fem-sg3 decreased fastest with highest change in slope among the same types, which means it has highest efficiency within three sgRNA.
Figure5: Relative fluorescence intensity of different sgRNA for femA
(represent the sensitivity of different sgRNA)
Besides, we test the reaction of the sgRNA in different concentration of bacteria liquid. By this, we can find the minimum detectable concentration, and get the sensitivity of our test kit. The higher gradient means the higher efficiency of cleaved the plasmid containing the target sequences.
At the point that relative fluorescence intensity gets 1000000, human eyes could identify the change in the light intensity. Therefore, the line of relative fluorescence intensity of different sgRNA that get the 1000000 in least time would represent the most effective sgRNA. We find out that sgRNA1 in femA group in GbcA group is the most sensitive sgRNA.
Learn 1
Our experimental results showed that our DETECTR system is reliable and our product can be used to detect Staphylococcus aureus. When the target gene femA was detected by the sgRNA, Cas12a was activated and cleaved them. We verified the efficiency and sensitivity of the sgRNA by gel electrophoresis and using fluorescent probes. We found that Cas12a could recognize and cleave the target gene effectively. And sgRNA was sensitive.
Construct design and build 2
pUC57-GbcA: BBa_K4926003
We designed a plasmid containing gbcA. The DNA fragment is from the genome of Pseudomonas aeruginosa to imitate the bacteria. In order to construct our plasmid, we let the company synthesize the DNA fragment GbcA, which was inserted into pUC57 plasmid.
Figure 6. pUC57-GbcA in DH5αα
Same with pUC57-femA, we let the company synthesize the fragment and verify the plasmid presented in the E.coli DH5α by electrophoresis. As shown in Figure 6 and 7, we got the bacteria containing the plasmid and lanes which length were same with GbcA.
Figure 7. Bacteria PCR of GbcA.
Function test 2
Figure 8. Graph of efficiency comparison of sgRNAs
We also used T7 in vitro transcription kit to obtain the sgRNA of GbcA. We mixed Cas12a, sgRNA and the plasmid. We tested the fluorescence of the sgRNA after incubation. We can see all the two sgRNA shows decreasing slope, all of them successfully react with the plasmids, which means our experiment succeed. The gbc-sg1RNA has highest efficiency.
Figure 9: relative fluorescence intensity of different sgRNA for GbcA
(represent the sensitivity of different sgRNA)
Besides, we test the reaction of the sgRNA in different concentration of bacteria liquid containing pUC57-gbcA. We find out that the sgRNA2 in GbcA group is the most sensitive sgRNA.
Learn2
This system also could be used to detect the Pseudomonas aeruginosa. When gbcA was detected by the sgRNA, Cas12a was activated and cleaved them. We could detect the bacteria by gel electrophoresis and using fluorescent probes.
On the whole, we have innovatively applied CRISPR/Cas technology to our diagnostic kit, expanding the scope of its application. Combined with simple heating steps, our kit allows for fluorescence readouts that are easily observable. In conclusion, our product is highly feasible and addresses many of the pain points and challenges associated with existing mainstream detection methods. It offers a new and promising solution for microbial detection.