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Our team is dedicated to modifying the original Chitinase through methods of synthetic biology, aiming for the enhancement of its binding ability to degrade Chitin in the fungal cell wall, which kills the fungus and achieves the purpose of treating skin diseases. During the experiments and the design and implementation of Human Practice and Education, we have summarized a few points that may be found useful for future iGEM teams, as follows:

Taking into account the matter of biosafety, our team initially used E.coli 1917 as our expression vector. Crediting its completely non-toxic property, E.coli 1917 could act on human skin directly to express chitinase. However, our team later recognized problems of this method regarding gene safety. Directly applying E.coli rapture to the skin may cause leakage of internal genes, which accounts for potential risks. Thus, we switched to using E.coli BL21, expressing chitinase in factories and purifying. Moreover, the expression ability of BL21 is much stronger than that of 1917, which could help reduce production costs.

The plasmid we obtained contains promoter, ribosome binding site (RBS), target fragment, and terminator. Taking into consider the universality of regulatory conditions, we chose to use Lac Promoter. It promotes transcription in the absence of Lacl and CAP proteins and inhibits transcription by binding to Lacl and CAP proteins in the presence of two sites. In order to control the transcriptional progress of E. coli BL21 better, we choose to add allolactose mimetic (IPTG) as the predetermined period to suppress Lacl protein and induce transcription. Future iGEM teams could choose to apply Lac Promoter for prokaryotic expression and optimize the expression efficiency by adjusting the IPTG concentration. The RBS we used is BBa_B0034, which has a medium to high strength; the terminator we used and worked best for us is BBa_B0015, a double terminator composed of BBa_B0010 and BBa_B0012.

Part of the fragment

Terminator

Our double terminator

Due the poor water solubility of Chitin, we prepared colloidal chitin. Specific experimental steps could be found in the following link:

link for experimental part

We used 3,5-dinitrosalicylic acid to measure chitinase activity. The theory is as follows: chitinase hydrolyzes chitin to produce N-acetylglucosamine, which reacts with 3,5-dinitrosalicylic acid to yield a red-brown compound with an absorption peak at 5400nm. We prepared the standard system at 540nm to measure the absorbance value, which reflects the degree of Chitinase enzymatic activity. For further details, please refer to the experiment steps provided in the following link:

link for experimental part

As mentioned earlier on this page, we choose to express chitinase in the factory and then purify it. To meet this requirement, a lysis system is necessary to destroy the E. coli BL21 cell wall. Our cleavage system contains arabinose promoters, BBa_B0034 RBS, SRRz cleavage genes, and BBa_B0015 terminators. The cleavage system is regulated by adjusting the concentration of arabinose, breaking the cell wall to release chitinase.

After analyzing our data from the experiment, the obtained the optimum pH value and temperature for the modified chitinase, which is highly universal. It can work under normal human body temperature and pH and it does not cause side effects to the skin. This could be used for references for future teams.

Our project provides numerous valuable suggestions and data for future teams: We should avoid gene leakage during the process of producing drugs that would be applied on human body; we could use BL21 in factories to express proteins with a suitable lysis system and remove endotoxins; we provided some procedures on how to prepare chitin Colloid and method for measuring chitinase activity; we identified good promoters, RBS and terminators for prokaryotic expression; we obtained the optimum pH and temperature of modified chitinase.