• Project Description
• Overview
• Background

• Ag43 Secretion System
• Overview
• Design
• Experiments
• Result
• Conclusion

• Other Systems
• His-tag Inactivation System
• Co-expression System
• Discussion

• Future Work

• Modeling

Project Description

Overview

Antimicrobial peptides (AMPs) have wide range of toxicity against microbes. Even if we hope E. coli to produce AMPs, it would be challenging task for them to do so because AMPs are toxic to the host bacterial cells. Thanatin, which is one of the AMPs derived from shield bug, is not an exception. It has wide-range of antimicrobial activity against bacteria, archaea, fungi and viruses. It is a short polypeptide composed of 21 amino acid residues.

Mass production of thanatin would be beneficial for human being, since AMPs could take the place of antibiotics in the future. So, we iGEM HokkaidoU Japan planned to produce thanatin using E. coli.

It is known that modification of thanatin’s C-terminal residues inactivates thanatin. So, we made thanatin tandem-multimer. In this thanatin-multimer, one thanatin covers C-terminus of neighboring thanatin and inactivates it. Using this method, we succeeded in losing activity of thanatin without killing its host cells. This means that we made epochal method to mass-produce host-toxic peptides.

Next, we need to collect thanatin. In order to collect thanatin easily, we utilized Antigen43 (Ag43). Ag43 is a member of autotransporter proteins. It has alpha-domain and beta-domain. Alpha-domain is called “passenger”, which is translocated from periplasmic space to the surface of cell through beta-domain. Beta-domain has unique Beta-barrel structure. This beta-barrel is a pathway for translocation of alpha-domain. So, we replaced the alpha-domain with thanatin-multimer. This could realize the secretion of thanatin. And then, monomerization of surface-displayed thanatin would make thanatin antimicrobial active.

This thanatin secretion system using thanatin-multimer and Ag43 beta-domain could be a big advance towards mass-production of AMPs. Besides from this system, we tried several other systems, which we ended up in finding out it was not suppressive enough, and killed out the host cells. We also designed AMP production system using Lactobacillus casei.

AMPs have wide range of toxicity against microbes. Even if we hope E. coli to produce AMPs, it would be challenging task for them to do so because AMPs are toxic to the host bacterial cells. Thanatin, which is one of the AMPs derived from shield bug, is not an exception. It has wide-range of antimicrobial activity against bacteria, archaea, fungi and viruses. It is a short polypeptide composed of 21 amino acid residues.

Mass production of thanatin would be beneficial for human being, since AMPs could take the place of antibiotics in the future. So, we iGEM HokkaidoU Japan planned to produce thanatin using E. coli.

Summery

• Even we tried several ways to inactivate AMPs again and again, we failed to get transformant that have AMPs gene again and again because of its strong toxicity. However, we succeeded in making AMPs inactivation and production system using a kind of outer membrane protein, Ag43.
• We made new biobrick parts that have functions to produce host-toxic peptide, AMPs.
• We improved preexisting biobrick part BBa_K759001 to secret a kind of AMPs, thanatin. The parts are BBa_K1714000, BBa_K1714005, BBa_K1714006, BBa_K1714007.

Background

Until now, more than million species of insects have been found on Earth, and it is said that insects occupy about 70% of animals living on Earth. Though they are most prospering animal on land, unlike vertebrates, they do not have acquired immunity using antibodies. Instead, insects developed innate immune system. After the long history since the first insect had appeared on Earth, it is said to have been very rare to have insects die out due to microbes getting resistant of their immune system. Insects were able to have this success thanks to antimicrobial-peptides, or AMPs.

Antimicrobial peptides (AMPs) are, as its name says, peptides that attack pathogenic microbes. They are usually several tens of amino acid residues long, and are positively charged since they usually have basic amino acids. This positive charge enables the peptides to interact with cell membranes, and makes a hole. Contents of the cell outflow form this hole and the cells get killed.

AMPs are found in wide range of spices, including humans. Many of these kills wide range of microbes, including fungi, gram-positive and negative bacteria.

Recently, multidrug-resistant microbes have been a big program. From the fact that using AMPs, we could kill these bacteria, AMPs have been studied well for new kind of medicine.

Besides chemosynthesis, methods using genetic engineering have been a big topic for mass-producing AMPs. Program here is that because AMPs kill bacteria, it is difficult to synthesize AMPs using bacteria, since it kills itself. Many research have done to solve this program, including co-expression with lactalbumin family and expression using inclusion body, which both showed decrease in cell toxicity. (S. Tomisawa et al., 2013 ^[1]) There is also a research on alpha-defnsin, kind of AMP expressed from paneth cell of small intestine, which says that alpha-definsin have a selectivity to kill microbes that is not a resident flora. (K. Masuda et al., 2011 ^[2])

Using these facts, we HokkaidoU_Japan decided to produce two kinds of AMPs, thanatin and alpha-defensin, using Escherichia coli and Lactobacillus casei.

Reference

1. Tomisawa, S., Hojo, E., Umetsu, Y., Ohki, S., Kato, Y., Miyazawa, M., ... & Aizawa, T. (2013). Overexpression of an antimicrobial peptide derived from C. elegans using an aggregation-prone protein coexpression system. AMB Express, 3(1), 45.
2. Masuda, K., Sakai, N., Nakamura, K., Yoshioka, S., & Ayabe, T. (2011). Bactericidal activity of mouse α-defensin cryptdin-4 predominantly affects noncommensal bacteria. Journal of innate immunity, 3(3), 315-326.

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