Description | Thinker-Shenzhen

1. Origin

Soon to be the core of our team reserves discussing the health of modern people, not as the twenty-first century, although the level of healthcare has been greatly improved, and extremely dangerous diseases have been well controlled, but there are many

people are in sub-healthy living conditions, either mentally or physically, skin diseases is one. Skin diseases can result from many factors, such as fungi, bacteria, allergens, etc., with fungal skin diseases being one of the more common ones.

There are many kinds of fungal skin diseases, such as beriberi, candida, ringworm, etc. This superficial fungal infection not only makes the infected person's skin itchy and unsightly, but may also turn into a deep fungal infection if left untreated, which can seriously affect health. At the same time, the prevention and treatment of fungal skin diseases can place a considerable burden on the public finances.

Thus, it is imperative to find an efficient and innovative method that can cure fungal skin diseases.

dermatomycosis

The burden of Fungal Diseases

There are numerous dermatological drugs available on the market already. Terbinafine is a common synthetic arylamine antifungal drug that can cure tinea pedis by inhibiting the synthesis of ergosterol by inhibiting the enzyme squalene cyclooxygenase, which in turn increases the permeability of cell membranes causing the cells to lyse and die. However, this chemical drug has the potential to spread to other parts of the body, sometimes causing symptoms such as hepatitis, autoimmune reactions, and abnormal sensation. Another typical triazole drug is itraconazole, which reduces ergosterol production by inhibiting cytosolic pigment P450 oxidase, increasing cell membrane permeability and eventually causing cell lysis. Itraconazole is relatively well tolerated, but also has side effects including nausea, vomiting, etc. In addition, this chemical class of drugs is very expensive, the treatment cycle will probably cost more than a thousand dollars, which is a great burden, some patients will stop the drug early, which will increase the risk of fungal regeneration in the body.

Itraconazole

Through our efforts, several members of our team successfully contacted several faculty members at Lanzhou University and had discussions related to fungal skin diseases. Based on our understanding, we found that most common medications have a diminishing effect and contain chemicals that make it impossible to avoid some side effects.

So how can we avoid the use of chemical drugs?

After our team members' communication with the teachers several times, through the study of the fungal cell structure, we finally decided our targets - the chitin in the cell wall.

2. Chitin

Chitin is a nitrogenous polysaccharide substance, consisting of approximately 8000 N-acetylglucosamine aggregates, commonly found in fungal cell walls and give strength.We believe that by degrading chitin in the fungal cell wall we can destroy its structure and achieve the goal of killing the fungus and successfully treating fungal skin diseases. Therefore, we have reviewed a lot of information on how to degrade tributyrin.

According to our understanding, chitin can be turned into cellulose by the action of deacetylase and deaminase, can be hydrolyzed by lysozyme, and likewise can be degraded by chitinase. Among these three methods, we decided to use a tributyl hydrolase to break the β(1,4) glycosidic bond in tributyrin to degrade tributyrin.

Chitin in the cell wall of fungi

Use deacetylases and deaminases to degrade chitin

3. Improvement

We determined the solution and then used a commercially available chitinase to degrade the fungus. The chitinase hydrolyzed the chitin to produce N-acetylglucosamine, which further reacted with 3,5-dinitrosalicylic acid to produce a brownish-red compound, which had an absorption peak at 540 nm, and we determined the chitinase activity by measuring the change in absorbance. We were surprised to find that the results were much lower than we expected. We were surprised to find that the results were much lower than our expectation. We were puzzled by this situation, did our thinking go wrong at the beginning, and the tributylase could not degrade the tributylin in the fungal cell wall well? So, we turned our attention back to the fungal cell wall structure and realized that the situation was not as simple as we thought.

The chitin in the fungal cell wall exists in the inner junction with the cell membrane, and it is difficult for chitinase, which only has one catalytic domain, to bind and degrade the chitin in the cell wall, so how to break through the outside of the cell wall and bind the chitin is the most important problem we need to solve.

Chitin in the inner cell wall

After investigating the structure of many titinases, we found that some titinases have a chitin-binding structural domain at the nitrogen- or carbon-terminus, which is a carbohydrate recognition binding module that, although not degradative, can bind to chitin and send it to the intermediate chitin catalytic section for degradation, which in theory would increase the degradation efficiency of titinases and better satisfy our need for rapid fungal killing.

Schematic diagram of working principle

Once we renewed our ideas, we started working on the product design. We understood the catalytic structural domain of Streptomyces alfalfae tributylase, the nitrogen-terminated tributyl binding structural domain of Bacillus cereus tributylase, and the carbon-terminated tributyl geometric structural domain of Bacillus thuringiensis, respectively. To avoid biosafety issues, we designed and obtained the gene sequences of the modified chitinases directly using DNA synthesis techniques. The addition of the chitin-binding domain to the ends of the catalytic section of our chitinase would theoretically greatly enhance the binding capacity for efficient fungal killing.

Improved Chitinase

Before and After Gene circuits

We used the chassis microorganisms E.coli DH5α and E.coli BL21, and a large number of titinases were obtained by transforming the obtained DNA sequences on plasmids such as E.coli DH5α for biological replication and then transforming them into E.coli BL21 for protein expression.

4. Cracking System

Considering the biosafety issue, in order to release large amount of chitinase and prevent contamination by gene leakage, our E.coli BL21 cannot be directly applied on human skin, so we choose to design a lysis system that can destroy the cell structure of E.coli in the factory, release the produced chitinase and purify it to make a high purity enzyme product to treat fungal skin diseases.

Our lysis system includes the L-Arabinose manipulator, BBa_B0034 RBS, S-rrz gene and BBa_B0015 terminator. The lysis gene is introduced into BL21 and a certain concentration of arabinose is put in to initiate the lysis process, and the perforin gene s product in the S-rrz gene changes the cell membrane permeability and forms a porous structure in the cell membrane so that the r and The transglycosidase gene r produces a water-soluble transglycosylase to break down the peptidoglycan in the E. coli cell wall; the rz gene produces a peptide chain endonuclease that cleaves the linkage between the peptidoglycan oligosaccharides and the cross-linkage between the peptidoglycan and the outer membrane of the cell wall. Eventually the E. coli BL21 cell wall breaks down, releasing a large amount of chitinase, and after a purification process to remove impurities such as endotoxin from the product, we obtain a high purity chitinase, which is then made into a drug to treat skin diseases.

Cracking System

5. Overview

In general, our project consists of two modules: DNA synthesis of chitinase target fragment with chitin-binding structural domain and SRRz lysis system to disrupt E.coli BL21 cell wall to release expressed chitinase, purify chitinase, remove impurities such as endotoxin, and make high purity active chitinase drug for fungal inhibition and treatment of fungal skin disease on the affected area, which is non-toxic and non-harmful. There are no biosafety issues.

Improved Chitinase

Cracking System

Reference:

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