Team:Jilin China/Protocol
Plasmid construction
The new plasmid constructed was based on the template, pgh. Two orf and one ori , which were selected from PMB-1 and related to gene duplication, were constructed into the plasmid. And then, a HU promoter was constructed to promote the expression of the gene encoding a histone-like protein. At last, vP3 was added into the plasmid. The construction of the plasmid was outsourced to the company. We improve it by adding the signal peptide (Tmp1 and Sec2) to help the secretion of apoptin protein and TAT to help the entering of apoptin into the cells. We added a linker between TAT and apoptin. We designed three plasmids (Tmp1-TAT-Linker-apoptin,Sec2-TAT-Linker-apoptin,TAT-Linker-apoptin) and have them synthesized by company.
Plasmid was transformed into E.coil
The competence DH5α and plasmid were kept on ice. 2 μl plasmid was put into 50 μl DH5α and kept on ice for 30min. Heat shock was performed the at 42℃ for 90sec. Then put the tube on ice quickly for 2min.Then pour 25μl DH5α that had transformed plasmid on the plate and spread them evenly by spreader. Then seal the petri dish by sealing film and culture it in 37℃ for overnight.
