Based on CRISPR pathogenic microbial detection technology was developed in recent years, and the DETECTR system, which is based on CRISPR-Cas12a, was applied for pathogenic microorganism detection. In order to verify if there are related parts, we searched the iGEM Biological Parts library and picked BBa_K2644101. This is a biological part submitted by iGEM18_TJU_China in 2018, and the team provided the DNA sequence of FnCas12a, and they measured the effect of ions on FnCas12a’s cleavage activity. Our team developed a reaction platform to detect pathogenic microorganisms, such as salmonella and shigella, adding data from in vitro DETECTR reaction system. This information can be a good reference for future iGEM teams working on in vitro DETECTR reaction systems.
The gene fragments ipaH and invA are amplified from salmonella and shigella genomic DNA, we synthesized these DNA fragments and insert them in the pUC57 vector. Next, we mixed the FnCas12a protein with sgRNA corresponding to the two genes, after they formed a complex, then we added these plasmids and buffer. Finally, we did gel electrophoresis to verify if the in vitro reaction system worked well.
FnCas12a, which is amplified from Francisella novicida, is a new class II family of CRISPR-Cas RNA-programmable endonucleases with unique features that make it a very attractive alternative or complement to Cas9 for genome engineering.
We send the company to synthesize the DNA fragments of ipaH and invA, and inserted them into the pUC57 vector, the sequenced data showed that the plasmids which were provided by the company were successfully constructed (Figure 1). 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, then we extracted the plasmids.
Figure 1. the sequencing data mapped to the DNA fragments. 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 2).
Figure 2. 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.
This is a plasmid including the DNA sequence of FnCas12a protein that was integrated (Figure 3). This sequence will recognize the sgRNA sequence and form a complex that can identify the targeted DNA sequence of the bacteria we want to identify. This plasmid contains Kana+ resistance and could be used to screen for transformed colonies. The expression of FnCas12a could be induced by IPTG.
Figure 3. the plasmid map of pET28a-FnCas12a
We designed this plasmid by inserting ipaH DNA fragment into the plasmid pUC57 with a T7 promoter and terminator in front and after the sequence. This plasmid is used to obtain the gene fragment of Shigella which will bind with sgRNAs and recruit cas12a protein to cleave the DNA fragment of it.
In this part, the 16S DNA fragment is amplified from H.pylori genomic DNA. 16S ribosomal RNA sequences have been used extensively in the classification and identification of Bacteria and Archaea. The comparison of almost complete 16S rRNA gene sequences has been widely used to establish taxonomic relationships between prokaryotic strains, with 98.65% similarity currently recognized as the cutoff for delineating species. The DNA sequence of 16S is conserved, so it could also be used to distinguish H.pylori from other pathogenic microorganisms. This plasmid is used to obtain the 16S DNA fragment which will bind with sgRNAs and recruit cas12a protein to cleave the DNA fragment of it.
