Many cosmetics that contain high water content and abundant nutrients are susceptible to microbial contamination.Staphylococcus aureus and Pseudomonas aeruginosa are two types of pathogenic bacteria that can be found in certain cosmetics.
We designed a swift and straightforward detector to detect femA and GbcA, which were expressed in Staphylococcus aureus and Pseudomonas aeruginosa, respectively. This detector is based on the CRISPR/Cas12a-based DETECTR system, which is fast, accurate and easy to operate.
We designed and ordered three plasmids, pUC57-femA, pUC57-GbcA and pET-28a(+)-FnCas12a-6His. from other company laboratories. Genes femA and GbcA are amplified from the genome of Staphylococcus aureus and Pseudomonas aeruginosa respectively. In order to construct our plasmids, we let the company synthesize the DNA fragments, femA and GbcA. And these two fragments were inserted into pUC57 plasmid. pET-28a(+)-FnCas12a-6His was obtained from the company laboratories.
We transformed the pUC57-femA and pUC57-GbcA into the E.coli DH5α.As shown in Figure 1, we obtained the E.coli DH5α with target fragments of femA and GbcA. To verify whether these two plasmids presented in the E.coli DH5α, we performed TAE agarose gel electrophoresis. As shown in Figure 2, we got lanes which length were same with femA (1105bbp) and GbcA (646 bp). And we confirmed that these two plasmids were in the E.coli DH5α. (Figure 2).
Figure 1. pUC57-femA/pUC57-GbcA in DH5α
Figure 2. Bacteria PCR of femA and GbcA.
The corresponding genes of sgRNA of femA and GbcA were obtained by PCR (isothermal amplification) as DNA templates. We verified that the sgDNA is the one we planned to target by checking the bp length (Figure 3).
Figure 3. DNA fragment of femA and GbcA in target sgRNA
We then used T7 in vitro transcription kit to obtain the sgRNA of femA and GbcA. We also checked whether the sgRNA underwent a successful transcription by taking its DNA template as negative control. As shown in figure 4, since sgRNA of both femA and GbcA is under 100bp, we confirmed that the T7 in vitro transcription was successful.
Figure 4. femA and GbcA sgRNA and DNA template
We transformed the pET-28a(+)-FnCas12a-6His expression 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.
As shown in Figure 5, the size of the LbCas12a protein is between 100kDa and 160kDa. Hence we can see that the protein was successfully induced to express.
Figure 5. SDS-PAGE analysis of LbCas12a protein
Figure6. Graph of efficiency comparison of sgRNAs
After the testing of the fluorescence of the sgRNA after incubation, we draw this graph to show the efficiency of the different sgRNAs. In the graph, 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 gbc-sg1RNA and the fem-sg3 decreased fastest with highest change in slope among the same types, which means they have highest efficiency.
Besides, we tested the reaction of the sgRNA in different concentration of bacteria liquid containing plasmids pUC57-femA and pUC57-GbcA. By this, we can find the minimum detectable concentration, and get the sensitivity of our test kit.
Below in Figure 7 and Figure 8 are the graphical representations of the sensitivities of the five sgRNA: The vertical axis represents the relative intensity, which means the change in the intensity of the fluorescence. Therefore, the higher gradient means the higher efficiency of cleaved the plasmid containing the target sequences.
Figure7: Relative fluorescence intensity of different sgRNA for femA
(represent the sensitivity of different sgRNA)
Figure8: Relative fluorescence intensity of different sgRNA for GbcA
(represent the sensitivity of different sgRNA)
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. And by analyzing the 1ul and 2ul, we can find the sensitivity of each sgRNA. After the analyzing, we find out that sgRNA1 in femA group and the sgRNA2 in GbcA group is the most sensitive sgRNA.
In conclusion, each sgRNA has their own advantages and disadvantages. Therefore, we would not focus on any single type of sgRNA to make our product. We will try to mixed these sgRNA together in a specific concentration ratio to get the highest efficiency and sensitivity in detect the pathogen.
There are multiple pathogens that will lead to the infection in the cosmetics. In this project, we only designed the detector of two kinds of most usual pathogens. In the future, we can design more sgRNA for a larger detection range in our test kit. Besides, we can test different concentrations and combinations of the sgRNA and find the highest efficiency for sgRNA. Also, we will use other instruments like Cas13 and Cas 9 proteins for other pathogens in addition to the bacteria. Additionally, we can also design a test kit that can be applied to other conditions like food testing, water testing, or even air testing. As a result, with improved or multiple solutions for detecting, our test kit can be more useful and convenient.