BEST PARTS COLLECTION
Functionally, this year, our parts are mainly divided into 5 parts. The first part is divided into the system construction part, for which we designed the plasmid containing E gene to construct the RPA system and the CRISPR Cas12a system. The second part is the RPA amplification part, and we designed the primers required for RPA amplification.
The third part is the part of the CRISPR Cas12a, where we designed the sgRNA to recognize the target sequence and to activate the trans-cleavage activity of the CRISPR Cas12a protein.
The fourth part was a sample simulation part, and we respectively constructed plasmids containing cfDNA of Echinococcus and DNA of Schistosome, to simulate clinical samples and to detect the specificity of our system. The fifth part is the cost control part, in which we worked in cooperation with the NUDT_CHINA, where NUDT_CHINA designed a plasmid that efficiently expresses the CRISPR Cas12a protein to control the experimental cost.
On the engineering loop, our project is divided into two parts: AND and OR.
Our AND partially consists of the RPA system and the CRISPR Cas12a system, and after repeated practical experiments, we have achieved the first multi-primer pair isothermal amplification and multiple sgRNA-guided CRISPR Cas12a detection in the iGEM project since 2020.
Since the cfDNA in the patient's blood often exists in small, randomly broken fragments, we hope to improve the true-positive rate by detecting multiple sequences simultaneously. Our OR part consists of 6 primer pairs and the sgRNA targeting the corresponding sequence, and the simultaneous detection of multiple sequences increases our true-positive rate from 40% to 70%.
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