Team:GIFU TOKAI/Human Practices
Human Practice
How we perform Human Practice
Getting suggestions from GeneFrontier
GeneFrontier Corporation (GFC) is a Japanese biotech company, which is one of the group companies of KANEKA Corporation. GFC is focusing on biopharmaceutical R&D support business with innovative products and services, especially in protein-based biologics including antibody. The company makes PUREfrex kit; a reconstituted in vitro Coupled Transcription/Translation Systems utterly different from an E.coli extract S30 system. We have got lots of technical information concerning the products and translation system of prokaryotic cells to perform our study.
They told us the fact that the rate-limiting process of transcription/translation in PURE system is not the process of initiation of translation in PURE system, not like the cells. In their proceeding research, it is the process of elongation step. From their point of view, our research may not be useful to produce enough amount of biological medicinal products. However, they also mentioned that it might be possible for us to contribute to clarifying the process of recycling system of ribosomes. In current biology, the recycling step is unknown. Ribosome Recycling Factor (RRF) is a potential factor that is possibly related to the process, but the exact function is still indeterminate. Besides, EF-G is now thought to be another factor that may work to release the ribosomes from mRNA. So they advised us that we should study the conventional model of the recycling step to improve our project. As by-products of our study and system, the study may make it clear that the real translation system.
We also asked how to assay the translation speed in cell-free system. In their method, they used real-time PCR machine and microplate reader to do so. Measuring the amount of sfGFP by real-time PCR can be used to decide the initial speed. Microplate reader can work for quantifying the amount of sfGFP at endpoint. Furthermore, they added the comment of RBS intensity. Changing RBS intensity may be useful to assay iVEPOP if the phenomenon occurs.
From their advice, we learned a new aspect of iVEPOP and our study. Also, we could consider the practical methods to evaluate the translation speed in cell-free system. We want to say thanks to GeneFrontier to help us with giving advice and suggestions.
Consulting with Prof. Yokogawa
Professor YOKOGAWA is a specialist of cell-free system and protein synthesis system. His recent interests come to tRNA and its chemical modification. Prof. Yokogawa and his colleagues invented the reconstituted cell-free system, PURE system (Y. Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa, K. Nishikawa, T. Ueda, Nat. Biotechnol. 2001, 19, 751–755;). From his experiences of cell-free system and RNA synthesis in vitro, he recommended that we utilize 2-o-methyl-RNA primer and mix more than 15 mM GMP in transcription mixture.
When linear RNA is transcripted from linear DNA, T7 RNA polymerase typically adds extra base at the end of 3’ end The possibility of the phenomenon is said to be approximately 50%. In our project, extra-base of the 3’ end may cause low efficiency of circularization and was a critical problem which was needed to be solved. Thus he advised us to use the modified primer. Modification of 2-o-methyl-RNA at second base from 5’ end of Reverse primer can prevent from happening nontemplated addition of nucleotides to RNA fragments. Besides, he also taught us adding an excess amount of GMP to transcription kit to incorporate GMP to 5’ end. In our strategy of RNA circularization, T4 RNA ligase 2 joins the RNA strand by a phosphodiester bond. It is necessary to have GMP, not GTP to make this possible from the point of the chemical reaction. Hence, we added GMP more than ten times the amount of GTP.
Our project was based on the future work of iGEM Gifu 2018. We considered the project of iVEPOP with Prof. YOKOGAWA. See the attributions of iGEM GIFU 2018. We are respectful to his passionate attitude toward us and synthetic biology. Without his support from essential advice like treatment of RNA to technical suggestions like the examples above, we could not make circular RNA in vitro. This human practice really contributed to our project.