Difference between revisions of "Part:BBa K4304002"

Revision as of 08:36, 27 September 2022

cas12a-opt

Cas12a

Contribution

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 sgRNA 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 Biological Parts library and picked BBa_K3136004. This is a biologic part submitted by iGEM19_Shanghai_HS_United in 2019, and the team provided a complete reaction system to detect the presence of Listeria monocytogenes. Our team developed a similar reaction platform to detect pathogenic microorganisms, such as Helicobacter Pylori, salmonella, and shigella, adding data from in vitro DETECTR reaction system.

What’s more, once the endonuclease activity of FnCas12a was activated, this protein could also cut the non-target DNA fragments. Based on this phenomenon, we employed DNA probes with the 6-FAM flag on its 5’-terminal. When there was sgRNAs of target genes were recognized by FnCas12a, the probes would also be cleaved, then we could detect the fluorescence.

We synthesized four DNA fragments amplified from the pathogenic microorganisms we chose, and insert them in the pUC57 vector. Next, we purified the FnCas12a protein and synthesized probes with the 6-FAM flag. Then, we mixed those materials and the reaction buffer to develop the in vitro reaction system, and we also changed the concentration of the target genes. Finally, we detected the fluorescence of the reaction system and measured the activity of FnCas12a (Figure 1).

Figure 1. The workflow of our project.

Sequence and Features