Team:HokkaidoU Japan/Description
Project Description
Overview
HokkaidoU Japan 2015
Project Description
Insects are one of the most prospering kinds of living thing on Earth. Surprisingly, they do not have complex acquired immunity like we humans do. Instead, they only have innate immune system. This suggests that innate immunity may have very high potentials. Indeed, main immune system of insects using antimicrobial-peptides (AMPs) is known to work toward wide range of microbes, since they affect on cell membrane. Good part of AMPs is that it has less chance of getting resistant microbes.
This year, we iGEM HokkaidoU decided to work with AMPs.
Our first project is to make antimicrobial paper using thanatin, a kind of AMP. Our plan is to express thanatin with cellulose binding domain (CBD) in Escherichia coli. Difficulty here is that thanatin expressed by E. coli works to kill E. coli. To avoid this, we are planning to use inclusion bodies. By making thanatin peptides insoluble by putting 10xHis-tag, the peptides goes into inclusion body and get deactivated. In order to secrete thanatin from the E. coli cell, we are planning to use Antigen 43 and thrombin cutting site.
Our second project is to make antimicrobial yogurt using Lactobacillus casei. To do so, we are planning to use AMP called α-defensin. It is known from the research that in rats, α-defensins do not work towards resident flora (Masuda et al., 2011). According to the research group, similar tendency can be said to humans. In order to secrete α-defensin, we are planning to use sec system of L. casei. By having L. casei to secrete GFP withα-defensin, we can check whether α-defensin is being made correctly, and whether we have the safe yogurt.
Reference
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.
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 dye 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
- 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.
- 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.
