Revision as of 11:28, 19 October 2016

Team:HokkaidoU Japan - 2016.igem.org

Team:HokkaidoU Japan

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We focused on possible application of self-assembling peptides (SAPs). SAPs have a self-assembling ability because they are amphiphilic peptides and contain hydrophobic and hydrophilic residues. These electric 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 a good biological material. 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 each other through disulfide bonds and may constitute a 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 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. We were inspired by this project and we thought about trying to construct circularized enzymes by using SAPs. Then, we hit upon constructing circularized multi-enzyme. Finally, we focused on these two projects.

Parts designing

We designed parts using Benchling tooland used 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. After receiving them,we cut them by restriction enzymes and ligated another parts. GC rich or repeating sequences are difficult for synthesizing. Actually, we were rejected synthesis of these sequences and we could not proceed our projects smoothly.

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

@@ Line 70: / Line 70: @@
 <br>
 <h1>Parts designing </h1>
-We designed parts using <a href="https://benchling.com">Benchling tool</a>and used free DNA synthesis gBlocks® Gene Fragments from <a href="https://sg.idtdna.com/pages/products/genes/gblocks-gene-fragments">IDT</a>. IDT gBlock DNA can synthesize 125~2000bp. Because some parts are over 3000bp, we ordered splitting parts with restriction sites. After receiving them,we cut them by restriction enzymes and ligated another parts.
+We designed parts using <a href="https://benchling.com">Benchling tool</a>and used free DNA synthesis gBlocks® Gene Fragments from <a href="https://sg.idtdna.com/pages/products/genes/gblocks-gene-fragments">IDT</a>. IDT gBlock DNA can synthesize 125~2000 bp. Because some parts are over 3000bp, we ordered splitting parts with restriction sites. After receiving them,we cut them by restriction enzymes and ligated another parts.
 GC rich or repeating sequences are difficult for synthesizing. Actually, we were rejected synthesis of these sequences and we could not proceed our projects smoothly.

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