Team:GIFU TOKAI/Description
Description
Description of our project
Description
We, iGEM GIFU_TOKAI, focus on mRNA and changing its topological form into circular to create a new method for mass-production of protein in cell-free system this year. In the current research of circular RNA (circRNA) for protein production, expressing tandem-repeated protein was generated by circRNA without a stop codon. It shows circRNA has a potential ability that it can skip the rate-limiting process of the central dogma of molecular biology, binding ribosomes to mRNA. However, with conventional circRNA, functional protein cannot be translated because protein aggregation quickly occurs. Therefore, we decided to use translation-coupling system, which is found in operons of bacteria to produce monomer protein from circRNA. By applying it to circRNA, ribosomes repeat translation-coupling phenomenon in circRNA and are expected to express monomer protein. Our final goal is to produce functional proteins such as antibodies more efficiently and cheaper in cell-free system to provide medicaments consistently.
Inspiration
Various protein drugs, including antibodies for each disease, were found, and they are used for remedy in recent years. In very near future, we will need the antibody-drug more and more, but we cannot store almost all of them because it is unstable. Also, it is challenging to synthesize a large amount of them. Therefore, we decided to construct a system to mass-production of monomer protein as our first trial. To make it, we focused on cell-free system and circular RNA. As one example of the cell-free system, there is PURE system. PURE system is a kind of cell-free system established in 2016. The most prominent feature of PURE system is reconstruction type. The system contains only necessary factors for expressing, such as ribosomes. So, we can synthesize more clear proteins and purify more easily. It means that pharmaceutical companies can cut the cost, low the price of protein drugs.
Furthermore, the reaction is more stable than the conventional method. It is like a chemical reaction rather than a biological reaction. We also focused on another type of cell-free system like myTXTL. One of the problems of PURE system is its low efficiency of translation compared with other cell-free systems. We decided to use both of them to know which type of the cell-free system is fit in our project.
We also have our eyes on circular RNA. Translation reaction is subdivided into several steps, including initiation, elongation, termination, and ribosome recycling, initiation represents the rate‐limiting step. The rate-limiting step of translation is the step where ribosomes adhere to RNA. In circular RNA, once the ribosome is bound, it does not leave, and the rate-limiting step can be skipped, so it generates much protein faster than linear RNA. In the research of Perriman and Ares in 1998, they succeeded in expressing tandem-repeated GFP from circular RNA and showed it can be a revolutionary solution for a problem of the lack of expression power in the cell-free system since circular RNA can generate a large amount of protein compared with linear RNA. We set our goal of research to synthesize functional monomer protein a lot.
Besides, with the growth of the field of next-generation sequencing, unknown circular RNA is now being found in vivo. Even this research utilizes the logic of synthetic biology to solve the problem of the real world; our research is the latest in RNA engineering. Circular RNA is more stable than linear RNA because it is not decomposed by exonuclease in cells. Further applications would follow this project.