iGEM HokkaidoU_Japan 2015  Masaya Mitsumoto, Yuri Sakai, Tamaro Sakurai, Koya Ito, Naoki Onoda, Hiroki Ono, Satoshi Fukuda, Mami Tanaka, Miho Toyooka, Ruka Nishimura, Kazuhiro Mimata, Miku Fujita, Yukino Shibata, Osamu Horiguchi,  

Result Ⅰ  

   

   

Fig. 4 Growth curves of E. coli expressing different number of thanatin tandemmultimer. We measured temporal change of OD600 and draw growth curve. (a)Thanatin-monomer, (b) Thanatin-dimer, (c) Thanatin-trimer, (d) Thanatin-tetramer,(e) Growth curves of 5 transformants. As negative control, we also draw growth curveof E. coli containing only Ag43 β domain.

Fig.3B  

Fig.3A  

Fig.6  

Abstract  

Conclusions  ✔  We found that multimerization and fusion with Ag43 is one of the useful methods to produce host-toxic antimicrobial peptide. ✔  The result of MIC test indicates that we succeeded in cleaving thanatin by AspN, collecting thanatin from bacterial suspension culture, and reactivating antimicrobial activity of thanatin. ✔  We found that increasing copy number of monomer thanatin affects increase of antimicrobial activity of it. ✔  We succeeded in efficient production of host-toxic polypeptide in E. coli.

Reference  [1] Seiichi Taguchi, Kanako Kuwasako, Atsushi Suenaga, Miyuki Okada and Haruo Momose, Functional Mapping against Escherichia coli for the Broad-Spectrum Antimicrobial peptide, Thanatin, Based on an In Vivo Monitoring Assay System, J. Biochem. 2000, 128:745-754

Biodevice & System  

Fig. 3a Design of thanatin-4mer secretion system. Thanatin-4mer are inserted between signal peptide (S.P.) and Ag43 β domain. This biodevice also contains inducible promoter; PBAD, RBS and double terminator (dT).

Fig. 3b Activation process of thanatin. Cleavage by AspN will inactivate secreted thanatin. Multimerized thanatin do not have intense toxicity, but on being monomerized, it gets toxicity.  

Goals     1. Efficient Mass-production of host-toxic AMPs using E.coli.  2. Expression of AMPs as inactive form and collection of them after reactivation.  3. Innovation of a new method of AMPs production using Ag43.  

Result Ⅱ  

Fig. 5 Antimicrobial activity assay of thanatin. Growth inhibition of E. coli DH5α was assayed by adding 20 µl of serial dilutes containing thanatin into 80 µL of PB containing bacterial cells (cultivated to OD600 values of 0.4 and diluted 100,000 folds).  

Human practice  

Japanese consumers tend to avoid GMOs without any specific reason and evaluate them unfairly without considering the benefit of them. We thought that it is a very big program since GMOs could be a solution for upcoming food crisis expected sometimes in the future. And we conducted a survey to research the reorganization of GMOs among Japanese consumers.   As a result of survey, we made sure that

many people have negative impression of GMOs and GM Foods but they don’t have the evidence or knowledge of their negative impression of GMOs. So we explained what a GMO is and how it would act when it goes inside our body as approach to solution by some ways. In this year, we also participated in the online forum held by Tec_Guadalajara in Mexico as an activity of Human Practice or Collaborations. This online forum was held for shaping policy for our discipline of biotechnology/synthetic biology.  

Introduction  

 Structure of Thanatin  ・  derived from a shield bug  ・A short peptide composed of 21 amino acid residues.  ・It is known that modification of thanatin’s C-terminal residues inactivates itself.  →  We made thanatin tandem-multimer, one thanatin covers C-terminus of neighboring thanatin and inactivates it.  

 Characteristics of AMPs  ・a wide range of toxicity  ・AMPs affect to bacterial cell membrane, it is hard to get resistance to them  →Mass-production of AMPs would be expected.  BUT, even if we want E. coli to produce AMPs, it would be a challenging task because AMPs are host-toxic.  

 Structure of Antigen43 (Ag43)  ・Antigen43 (Ag43) has α-domain and β-domain.  ・The α-domain is translocated from periplasmic space to the bacterial surface through the β-domain.  ・The β-domain is β-barrel structure as a pathway for translocation of α-domain.  →We replaced the α-domain (a) with thanatin-multimer (b). By doing this, we succeeded in secreting the thanatin.  

楡法律事務所 　弁護⼠士 　宮永尊⽂文 はりきゅう 　キッズ・アンド・パートナーズ 株式会社シャネル化粧品技術開発研究所  

Sponsors  

Antimicrobial peptides (AMPs) have wide spectrum of antimicrobial activity. Since it is difficult for microbes to get resistance against AMPs, it has a potential to play a role as a medical drugs like antibiotics. So, efficient mass-production of AMPs is anticipated. However, since they are toxic to host bacterial cells, mass-production of AMPs using E. coli is difficult issue. For mass-production of AMPs, we iGEM HokkaidoU innovated a new method of AMPs secretion system by taking advantage of usage of autotransporter protein, Ag43. In this system, AMPs-multimer is fused to translocator domain of Ag43. And we succeeded in generating monomer AMP by treatment with endoproteinase, AspN. We measured its antimicrobial activity by measuring extent of reduction of optical density of E. coli, DH5α (MIC test). We could decrease AMPs’ activity by fusing them to translocator domain of Ag43. We also observed that increasing copy number of monomer AMP affects increase of antimicrobial activity of it. The results observed using MIC test of the AMP tetramer indicate that we could reactivated AMPs using combination of masking of antimicrobial activity by fusing it to Ag43 and regeneration of its activity by endoproteinase digestion. We succeeded in innovating new method for efficient production of AMP.