Difference between revisions of "Team:Gifu/Experiment-page"

PROJECT

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EXPERIMENTS

Making functional long chain protein

   In our last study, our long chain protein lost its function. As a reason, we had an idea that the protein lost its holding structure because it was much longer. Therefore, we aimed to synthesize functional long chain proteins by designing some linkers in this year.



Fig1. the role of linker

   The requirements for amino acids that can be good linkers are less of steric effects and strong in hydropathy. Glycine and serine fulfill this requirements. Glycine’ side chain has almost no steric effects. On the other hand, serine has some steric effects but it has powerful hydropathy because of its hydroxyl side chain. According to Mr. Yabe, because histidine has quite strong hydropathy, though it has big steric effects, it is possible that histidine can work as a linker.

Then, we designed some linkers [ see right ]. "GGSGGS", "GSGSGS", and "HHHHHH" were designed as linkers. We combined these sequence and inserted. The pattern of combination was shown right.

   Amino acid chains synthesized from circular mRNA are constructed from repetition in amino acid of target protein, amino acid from ribozyme and amino acid from RBS. In this experiment, we inserted linker sequences into inside of both splicing sites. Therefore, protein chains synthesized from circular messenger RNA that include linker sequences repeat linker sequence, amino acid from RBS, target protein, linker sequence and amino acid from ribozyme.
   Generally speaking, length of linker is required of one-forth of diameter of target protein. In the case of RFP, that condition is surely cleared by six amino acid, but it is unknown which linkers are appropriate in order to synthesize poly RFP protein. Therefore, we examined some types of amino acids and length of linker and choice the best linker in this experiment.

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developing the efficiency of circularization

About the complementary sequences at outside of both splicing sites

   In last year, we made a circular part by cloning splicing site from an intron of T4phage. There are the complementary sequences inside the splicing site in this intron. We thought that the sequence brings one splicing site close to the other one and ensures the reaction takes place.

   Therefore, we cloned splicing sites with complementary sequences in intron and made it into parts. Moreover, we made the device which express circular mRNA by using this part, and made its express in E.coli. Then, complementary sequences are included in outside of both splicing sites in RNA.

About the complementary sequences at inside of both splicing sites

   In case of designing complementary sequences at inside of both splicing sites, its sequence need including in a circular mRNA expressed in E.coli. We wanted to synthesize long chain proteins by making a circular mRNA translated semi-permanent. We had to design a complementary sequence which codes functional protein because the complementary sequence for inside was translated.

   In this study, we adopted two sequences. One is GGSGGS which works as linker, and the other is HHHHHH which becomes a histidine tag.