Team:HokkaidoU Japan/future

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α-Defensin Non-commensal Bacteria Killing System

Overview

Through eating foods, we take a lot of bacteria in our body. Of course, if such bacteria stay inside our body, they cause various illnesses. To protect our body, we have various immune systems like antibody, macrophage, and this year’s our subject; AMPs. While we defend our body from the invasion of bacteria, we live in symbiosis with commensal bacteria. These kinds of bacteria help us mainly in our intestine. They help us in digesting food, synthesizing nutrients, defending our body from non-commensal bacteria etc. Then, how do we live together with them even we have immune systems to eliminate bacteria? The secret was found in a kind of AMPs that human have. It is said that in our intestine, a kind of AMPs, α-defensin is secret and kill only non-commensal bacteria. About mouse α-defensin, this function has already reported (K. Masuda et al., 2011 ^[1]). About the human α-defensin, this function has not been reported, but by the same research group, similar function can be seen in human one. We thought if this AMP secretion system is injected to commensal bacteria, we can create new bio-device that kills non-commensal bacteria. We chose Lactobacillus casei as our chassis and cryptdin 4 from mouse intestine Paneth cells and human defensin 5 from human Paneth cells as α-defensins. Because L. casei is usually used for probiotics, we can use this bio-device for producing safer yogurt. The yogurt made of this bio-device is hardly contaminated by non-commensal bacteria. In addition, the bio-device doesn’t kill commensal bacteria, so we can use it with other probiotic commensal bacteria, like bifidobacterium bifidum (Fig. 1)

Fig. 1 This is an image of α-defensin selection system by commensal bacteria, L. casei.

Design

At first, because we chose L. casei as our chassis, the construct we use should have the replication origin and selection marker for L. casei. We used replication origin oripAM bata 1, replication protein repE and erythromycin resistant gene. Our construct also contains Sec dependent secretion system. Sec dependent secretion system is a kind of secretion pathway that L. casei has. Through this pathway, firstly proteins makes unfolded form by attaching Ffh, a kind of attaching protein to Sec secretion signal peptide.(Fig. 2)

Fig. 2 This is an image of protein folding inhibition through Sec dependent secretion pathway.

Thanks to Ffh, target proteins will be transported to a kind of outer membrane transportation protein complex, SecYEG complex. When the target protein passes through SecYEG complex, signal peptide is cleaved and the protein is folded and activated(Fig. 3).

Fig. 3 This is an image of protein folding and cleaving after passing through SecYEG complex.

So in this secretion system, while we can avoid inner toxicity of α-defensin, we can let L. casei secret it. In addition, downstream of α-defensin secretion system, there is GFP secretion system. So we can find α-defensin expression by seeing the green fluorescence of supernatant(Fig. 4).

Fig. 4 These images are the constructs for α-defensin non-commensal bacteria killing system.

Experiment

How to assay

First of all, we make sure that the constructs have correct sequence by sequencing. If we get the expected constructs, we transform it into L. casei (in this case, we used AHU1910)

1. Culturing the transformant.
2. Taking the supernatant and use it in MIC test.
3. [MIC test]

   Day 1

   1. Culturing E. coli JM109 in 2 ml of LB overnight.

   Day2

   2. Taking 80 µl of it and culturing about 1 hour in 2 ml of LB culture until the OD₆₀₀ become about 0.4.
   3. 100,000 fold diluting it with PB culture.
   4. Spreading 80 µl of them on LB plate and counting the number of colony.
   5. Making 20 µl of dilution series of supernatant and culturing 80 µl of diluted JM109 at 30 ℃ 120,000 rpm for 18 hours.

   Day3

   6. Incubating in refrigerator.
   7. Observing the appearance of colony and deciding effective concentration of supernatant.

Result

Even we tried colony PCR again and again, we could not find the colony which contains plasmid with complete α-defensin secretion system by wiki freeze. But now we are making transformant for making antimicrobial yogurt and we believe that making this bio-device gives us to think more about foods made of genetically modified organisms.

Reference

1. 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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