Team:KAIT JAPAN/experiments
EXPERIMENTS
Biobrick Design
- Cure allergic Rhinisis parts that are constantly expressed in the system
- The DNA sequence of the human-derived IL-10Reseptor gene STAT1 gene JAK gene was cited.
- In each DNA sequence, restriction enzyme sequence and primer sequence were designed like Biobrick part and were synthesized by IDT. (As shown below)
- We ordered other genes as well. However, as IL-10 receptor was difficult to synthesize due to a high proportion of GC, we converted the codon and synthesized it. Each gene was amplified with VF2 and VR by PCR and cloned by restriction enzyme treatment and ligation so as to be in the order of Plasmid 1.
- In the cloning stage, we transformed it into DH5α.
- Since we wants to express this part constantly, we inserted it into pCold plasmid and transformed into JM 109.
- Parts to express selectively in Cure Allergic Rhinitis System
- Like Plasmid 1, DNA sequence obtained by adding a membrane protein gene group (HlyD, HlyB, TolC) derived from E. coli and human-derived IL-12 gene to E. coli-derived HlyA signal peptide was cited. In each DNA sequence, restriction enzyme sequence and primer sequence were designed like Biobrick Part and were synthesized by IDT. Since we wished to use this Plasmid to control the expression, a human-derived GFAP promoter was used as the promoter. And since it was impossible to determine whether the HlyA signal peptide should be added before or after the gene, it was not possible to judge whether it should be added before or after the gene, so that the signal peptide was added before and after the signal peptide.
- In addition, Plasmid which IL-12 is changed to GFP, was also prepared to simplify functional tests.
GFP versions of plasmid were prepared by changing the position to which Hly A was added. 
- For demonstration,
- The two plasmids prepared are transformed into JM 109 and functional test is carrying out.
Demonstrate
- For demonstration,
Contact: kaitjapan@gmail.com Twitter:@KAIT_JAPAN
