Improvement

Overview

  Based on CRISPR pathogenic microbial detection technology was developed in recent years, and was applied to a series of pathogenic microorganism detection, and the DETECTR system is based on Cas12a, and once the target DNA-specific crRNA is detected, the Cas12a’s endonuclease activity would be activated and cleave both the target and non-target genes. In order to verify if there are related parts, We searched the iGEM Bio Parts library and picked up BBa_K4304002. this is the bio part submitted by Ykpao-iGEM22 in 2022. Our team developed a Cas12a system consisting of Cas12a and crRNA vectors that recognize and cleave miRNAs and release signals.
  Once the endonuclease activity of FnCas12a was activated, this protein c ould also cut the non-target DNA fragments. Based on this phenomenon, we employed DNA probes with the FAM flag on its 5’-terminal. When there were crRNAs of target genes were recognized by FnCas12a, the probes would also be cleaved, then we could detect the signals, and a color reaction occurs.

Introduction

 We constructed plasmids and inserted FnCas12 into the pET-28a plasmid vector. These constructed plasmids were contained in E. coli strains, and we serially inoculated plates containing the appropriate antibiotics on LB medium containing kanamycin resistance and incubated the vectors at 37°C overnight. (Figure 1)

Figure 1. Incubation of plasmids containing strains.
A. E. coli BL21 containing the pET28a plasmid. B. E. coli BL21 containing the pET28a plasmid.
C. pET28a plasmid after single colony culture. D. pET28a plasmid after single colony culture.

Purification of Cas12a protein and purified

                       

  Figure 2. Expression and purification of protein Cas12a. A. Incubation of Cas12a containing the pET28a plasmid. B. SDS-PAGE electrophoresis gels of Cas12a protein after IPTG induction at different concentrations. The Cas12a protein has a size of 130kDa.SDS-PAGE electrophoresis results showed that Cas12a protein was present in our collected solution at 130 kDa. It was found that 1 mmol/l IPTG was the optimal induction concentration by experiment. Therefore, Cas12a protein was expressed and purified with high quality. We tested the concentration of Cas12a protein by Bicinchoninic Acid Assay (BCA) using a SpectraMax i12x Multi-mode zymography with the absorption peak at 562 nm (Figure 3).

  Figure 3. Standard linear regression line of BCA method used to calculate protein concentration. Absorbance and calculated protein concentration of Cas12a. Through the curve values, we measured the absorbance of Cas12a as 0.2276 (L/(g.cm)) and the protein concentration (ug/ml) as 10.7549295, this result indicates that we obtained a sufficient concentration of Cas12a protein.


Testing the specificity of the Cas12a system after performing amplification experiments

Figure 4 Specificity results of miRNA-initiated isothermal amplification reaction


  From the figure, we can learn that the method we developed is characterized by high specificity. Different miRNAs can target different hairpin structures, thus triggering a thermostatic amplification reaction, which causes a clear difference between the chromogenic change and the blank and other kinds of miRNAs. It is expressed that after the HCR amplification reaction, different miRNA amplification products produce different degrees of signals after binding to the Cas12a protein. Proof of good method specificity Thus our experiments successfully verified the good specificity of the assay. the Cas12a protein and CrRNA can recognize and cleave miRNA amplification products to generate signals. Our experiment was successful.