We designed 5 plasmids: the FnCas12 protein expression plasmid, 16S, cagA, ipaH, and invA expression plasmids. Among them, the DNA fragments 16S and cagA are amplified from the genome of Helicobacter Pylori, and the gene fragments ipaH and invA are amplified from salmonella and shigella genomic DNA respectively.
In order to construct our plasmids, we let the company synthesize the DNA fragments, FnCas12 was inserted into the pET28a vector, and the fragments 16S, cagA, ipaH, and invA were inserted into the pUC57 vector. The constructed plasmids were contained in E. coli strains, we streak inoculated them on LB solid medium plates containing corresponding antibiotics, and incubate them at 37℃ overnight (Figure 1).
Figure 1. incubate the plasmids containing strains. 
Figure 2. 1.5% TAE agarose gel electrophoresis to verify the construction of oligo DNA containing plasmids.
We used TAE agarose gel electrophoresis to testify the presence of oligo DNA in the plasmid by performing PCR and then doing gel electrophoresis of the amplicons (Figure 2).
Our results show that a band of 200bp to 400bp is present in cagA, 16S, invA, and ipaH, but not in negative control (NC) lanes. Because oligo DNA has a size between 200bp to 400bp, our result supports the fact that the plasmids contain desired oligo DNA. The four plasmid transformations were successful.
We transformed the pET28a-FnCas12a expression plasmid into E. coli BL21(DE3) competent cells, and cultured at 37℃ overnight (Figure 3A). we inoculated a single colony into LB (Kana+) culture medium, incubated overnight, and then transferred the cultured medium into 1L fresh LB (Kana+) culture medium. We induced the expression of FnCas12a with IPTG when the OD600 was around 0.6-1.0, and cultured at 16℃ for 12h. Subsequently, we used nickel affinity purification to purify the acquired Cas12a proteins from other proteins in E. coli (Figure 3B).
Figure 3. Expression and purification of protein FnCas12a. Cas12a protein has a size of 130kDa. The SDS-PAGE electrophoresis result indicates that the Cas12a protein is present in the solution we collected at 130kDa, and not present in the nonspecific protein impurities. Thus, Cas12a proteins were expressed and purified with high quality.
Then, we tested the concentration of Cas12a protein by Bicinchoninic Acid Assay (BCA), using SpectraMax i3x Multi-Mode Microplate Reader with the absorption peak at 562nm (Figure 4).
Figure 4. BCA method standard linear regression line for calculation of protein concentration.
| Absorbance (L/(g·cm)) | Protein Concentration (µg/ml) | |
|---|---|---|
| Cas12a 1 | 0.2057 | 10.9358974 |
| Cas12a 2 | 0.1775 | 7.32051282 |
With this BCA standard curve, we measured the concentration of two samples of Cas12a protein, they are 10.9 µg/mL and 7.32 µg/mL respectively. This result indicated that we obtained a sufficient concentration of Cas12a protein.
In order to verify if FnCas12a we purified could precisely recognize and cut the target DNA sequence, we developed an in vitro reaction platform. Firstly, we obtained the sgRNAs through an in vitro transcriptional method and extracted the target sgRNAs fragments. Next, we mixed the purified FnCas12a protein, the sgRNAs, the corresponding plasmids containing DNA fragments, and the reaction buffer together. Then we incubated the reaction system at 37°C for 2 hours, and we verified the result by gel electrophoresis (Figure 5).
Figure 5. 1.5% Agarose gel electrophoresis comparing before and after cleavage of oligo DNA. After Cleavage is constituted of oligo DNA after cleavage by Cas12a protein and sgRNA. In contrast, NC (negative control) contains oligo DNA before cleavage only. Compared to NC, The oligo DNA band is significantly diminished after cleavage. This displays that the in vitro cutting experiment is successful.
To assess if our Cas12a-based system worked well, we designed a reporter system by ligating a 6-FAM at the 5’ terminal of the target DNA probes. Then we measured the fluorescence intensity using SpectraMax i3x Multi-Mode Microplate Reader every time the oligo DNA concentration increased, with the excitation of 485nm and emission wavelength of 507nm.
Figure 6. Fluorescence intensity at different concentrations of oligo DNA in simulated bacteria. The time between oligo DNA mixed with Cas12a protein and sgRNA system and measuring a sharp change in fluorescence intensity within 10 minutes. As shown in the graph, as the concentration of oligo DNA increased from 0 ng/μL to 4 ng/μL, the fluorescence intensity of the ssDNA fluorescent probes of the ipaH, invA, cagA, and 16S systems also increased significantly. The fluorescence intensity of a system with higher oligo DNA concentration is always higher than the fluorescence intensity of a system with lower oligo DNA concentration. Also, our results showed that the fluorescence intensity of the oligo DNA represent group is similar to or higher than the negative control (Figure 6).
Thus, the results indicate that our fluorescent detection experiment is successful. Cas12a protein and sgRNA can recognize and cut the oligo DNA probes and the fluorescence emission can be detected.
Figure 7. 1.5% TAE agarose gel electrophoresis to verify the amplified oligo DNA fragments.
The lamp is a novel nucleic acid isothermal amplification method, which is widely used in the rapid detection of bacteria, pathogenic microorganisms, parasites, and viruses. Compared with the currently widely used real-time qPCR technology, it requires four to six template-specific primers and DNA Polymerase with strand displacement activity, and the amplification is completed within 1 h under isothermal conditions; The corresponding detection dye can determine whether there is an amplification product. In order to make our detection kit more suitable for home portable use, we tested amplified oligo DNA with the lamp method, which is used as an alternative to the PCR amplification method. The experimental results show that there is our target band at the position of 500bp, indicating that the scheme of amplifying oligo DNA by the lamp method is feasible. This provides strong experimental data support for our development of portable detection kits.
