Team:HokkaidoU Japan/Overview

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Team:HokkaidoU Japan

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• Top
• Project
  • Overview
    • Project Description
    • Background
    • Proof of concept
    • Modeling
    • Achievements
  • MULTIMERIZATION
    • Overview
    • Methods
    • Results
    • Conclusion
  • FUTURE WORK
    • Circularization
    • Kill Switch
    • Transport
    • Aggregation
  • Extra Materials
    • Parts
    • Notebook
    • Protocols
    • Safety
• Human practices
• Collaborations
• Team
  • Attribution
  • Member

We focused on possible application of self-assembling peptides (SAPs). SAPs have a self-assembling ability because they are amphiphilic peptides, that is, peptides composed of hydrophobic and hydrophilic residues. These electrically charged amino acids interact with one another to form spontaneously antiparallel β-sheet in a physiochemical environmental condition.
In this project, we used one of SAPs, RADA16-I (RADARADARADARADA) and P11-4 (QQRFEWEFEQQ). Fig. 1. RADA16-I and P11-4 self-assemble under suitable physiochemical conditions
due to the polar amino acids and hydrophobic interaction and form β-sheet.

Fig. 2. Forming multiple GFPs ring (Self AssembRing)

RADA16-I has been established for 3-D culture of neural stem cells (NSCs) by creating nanostructures. P11-4 has been designed to form fibers at low pH and have a cytocompatibility.
Recently, SAPs have proved to be good biological materials. We investigated new application methods of SAPs through discussions. Finally, we decided to construct multi-enzyme-complex platform using SAPs and short linkers (SL). They are covalently linked with each other through disulfide bonds and may constitute circularized multiple enzymes (Self AssembRing).
In the future, we will construct a tool for making subunits of artificial multi-enzyme-complex by using this technology.

Self-Assembling Peptides

Recently, self-assembling peptides (SAPs) have been noted as biological nanostructures and hydrogel because SAPs are provided with biogenic suitabilities and don’t have cytotoxicity. SAPs have been used as a scaffold for cell culturing and drug delivering in tissue engineering and medical technology. For example, RADA16-I self-assembles nanofibrous forming hydrogel and is used for 3-D culture such as neural stem cells (NSCs), bones, cartilage tissues. However, SAPs are seldom used in iGEM, and the only past examples are ones such as EAK16-II (2008 iGEM Imperial College London) and RADA16 (2009 iGEM Slovenia). We need to construct new application methods and use them more often.

Enhancement of enzymatic activity

Improving enzymatic activities is needed to promote chemical reactions. Enzymatic activities can be enhanced by various ways including amino acid substitution, enzyme compartmentation, circularization, and multimelarization. For example, 2014 iGEM Heidelberg team constructed covalently linked circulated enzymes by using intein and extein to increase the enzyme stability. Having been inspired by their projects, we worked on circularized enzymes using SAPs. This led us to an idea of constructing circularized multi-enzyme. Finally, we focused on these two projects.

Parts designing

We designed parts using Benchling tooland employed free DNA synthesis gBlocks® Gene Fragments from IDT. IDT gBlock DNA can synthesize 125~2000 bp. Because some parts are over 3000bp, we ordered splitting parts with restriction sites, which were subsequently cut by restriction enzymes and ligated to other parts. GC rich or repeating sequences are difficult of synthesizing. Although we worked hard on the experiments, it is most unfortunate that the sequences proved to reject synthesis, yielding unsatisfactory results. However, most of the sequences turned out to be profitable.

Bronze

• We completed the team registration.
• We wrote team wiki.
• Poster and a talk for the iGEM Jamboree are ready.
• We described all attributions clearly.
• We created and documented Parts pages.
• We submitted DNA samples of our new BioBrick Parts.
• We filled out Safety forms.
• We filled out Judging form.

Silver

• We validated our new BioBrick parts.
• We collaborated with other iGEM teams (Gifu, Kyoto, Nagahama, UT-tokyo, METU HS Ankara, Heidelberg).
• We considered that prevalence of synthetic biology.

Gold

• We improved a previous part.
• We demonstrated a functional proof of concept of our project.

Proof of concept

[1] Riley JM, Aggeli A, Koopmans RJ, McPherson MJ, (2008) Bioproduction and Characterization of a pH Responsive Self-Assembling Peptide. Biotechnol Bioeng. 103(2): 241-51
[2] Kyle　S, Aggeli A,Ingham E, McPhersona MJ, (2010) Recombinant self-assembling peptides as biomaterials for tissue engineering．Biomaterials. 31(36): 9395-9405
[3] Prakash A,Parsons SJ, Kyle S, McPherson MJ (2012) Recombinant production of self-assembling β-structured peptides using SUMO as a fusion partner. Microbial Cell Factories. 2012, 11:92.
[4] Liang P, Xiong J, Zhao L, Xu Y, Zhao J, Liu Q (2015) Recombinant self-assembling 16-residue peptide nanofiber scaffolds for neuronal axonal outgrowth. Eng. Life sci. 15(1): 152-158
[5] Nune M, Kumaraswamy P, Krishnan UM, Sethuraman S, (2013) Self-Assembling Peptide Nanofibrous Scaffolds for Tissue Engineering: Novel Approaches and Strategies for Effective Functional Regeneration. Curr. protein pept. sc. 14: 70-84.
[6] Reed CD, Barnard GC, Anderson EB, Klein LT, Gerngross TU (2006) Production and puriWcation of self-assembling peptides in Ralstonia eutropha. Protein expres. purif. 46: 179-188.
[7] Zhang S, Behnke RE, Zhao X, Spirio L, PuraMatrix: Self-assembling Peptide Nanofiber Scaffolds. Tissue eng.