Difference between revisions of "Team:TJU China/Demonstrate"

In view of the current serious pollution problems, we focus on the pollution of heavy metal ions. Only by detecting heavy metal ions quickly and accurately can we prevent pollution in a timely manner. To this end, we started with a arsenic ion, combined with the achievements of the 2006 iGEM team (iGEM2006_Edinburgh) in order to construct a circuit dedicated to the detection of arsenic ions, which consists of Promoter J23104, ArsR Protein, Promoter ArsR, smURFP. We first ligated these fragments by overlap, then ligated them to the pKM586 plasmid by double restriction enzymes, and then transformed them into E.coli BL21. Since we think this loop can not be completed to meet our requirements, we want to make this loop more sensitive. Therefore, we noticed that dcas9 in the CRISPR-Cas system has an enhanced transcriptional effect, thus amplifying the effect of arsenic ions on the loop. In the plasmid of dCas9, we need to cut the two segments of the plasmid with BsaI enzyme, then connect the spacer we designed to target dCas9 to the corresponding gene, and then we import it with another plasmid E.coli BL21 to complete the enhancement of our arsenic sensing circuit by dCas9.

In order to reach a new gene detection in a high-throughput technique, CRISPR-Cas12a system is modified to chips which have been tiled with a layer of Janus (the hydrophobic protein designed by our team Tianjin in 2015) in advance. After our first step on the assembly of FnCas12a and crRNA was taken，we successfully tested sequence-specific cleavage activity on plasmid and trans-cleavage activity on ssDNA. With tremendous trails, we optimized cleavage protocol of both cis and trans cleavage. Last but not least, for achieving high-throughput detection on chips, we dried the Janus on the chip, then incubated Cas12a protein and crRNA complex, and finally, the fluorescence probe was cut and detected by fluorescence microscope. With the help of Janus, we were glad that higher fluorescence values were detected under the condition of a smaller amount of materials.

In order to realize the targeted delivery of sgRNA/Cas9 complex into cells, we make use of BODIPY, a kind of fluorescent dyes, to combine with sgRNA/Cas9 complex (RNP) and to deliver them into cells, since BODIPY can target nucleus itself[1] and can be observed as near-infrared (NIR) dye. Firstly, we constructed the template of sgRNA and completed the in vitro transcription of sgRNA using T7 promoter. We also expressed and purified Cas9 protein, and then we successfully tested the cleavage activity of sgRNA/Cas9 complex on plasmid. Then we combined BODIPY with sgRNA/Cas9 (BODIPY/RNP) and proved that our new complex had high in vitro cleavage efficiency. So we proceeded to the delivery of BODIPY/RNP into cells and found that BODIPY/RNP had better gene editing efficiency than RNP only and Liposome/RNP complex. After we succeeded in targeting nucleus, we tried to target not only nucleus but also mitochondrion. The plasmid we used was the pET-NLS-Cas9-6xHis plasmid, digested with Xba1 and Nhe1. In order to target mitochondria, we need to replace the NLS leader sequence on the plasmid with MTS (mitochondrial targeting sequence). We selected three MTS sequences, COX8a, SOD2 and ATP5. Since the MTS fragment is small and there is no suitable restriction site at both ends, we synthesized the MTS and the previous fragment. By annealing the two primers and then connecting the preceding fragment to the segment of Cas9 by overlap, we get the entire fragment. We then digest it withXba1 and Nhe1 and connect it with the vector.