Project

Circular mRNA -the world´s longest protein-

Project Description

   This year, the theme of iGEM Gifu is to synthesize a long chain protein with circular mRNA. It is a sequel to our theme of the last year.

   Here, we explain our theme of the last year. The circular mRNA is expressed in E. coli by using a mechanism of self-splicing. In td gene of T4 phage, there is the group 1 intron. A part of the RNA transcribed from the group 1 intron is a ribozyme which catalyzes a splicing reaction, and this RNA splices itself. The circular mRNA can be expressed in E. coli by cloning a domain which functions as ribozyme for splicing of the td gene, and putting it in the plasmid. This method is called “PIE method.” The circular mRNA without the termination codon sequence can be used to synthesize protein semi-permanently.

   We succeeded in expression of the circular mRNA, and synthesizing long chain protein last year. But the long chain protein synthesized in last year was not functional because its holding had lost shape. So, this year, we design a linker sequence and synthesize a functional long chain protein. On the other hand, the probability was just a few percent that the cyclization happens. It is thought that the ribozyme site of the splicing is far from the reactive site, and it lowers the probability. So we’ll design complemental sequence near the ribozyme site and the reactive site of splicing to make them accessible to each other, and raise the probability of the cyclization in the theme.