Team:TU Darmstadt/project
ChiTUcare
The Project: ChiTUcare
Here, we will present our project in a way everybody can understand it. After this REALLY simplified abstract, we should go a tiny little bit more into detail. Please have in mind that this part should still be about explaining the WHOLE project. One thing we could do but don't have to is to create boxes explaining the enzymes or subprojects when hovering over a highlighted text line such as 'chitinase'.
Chitin Synthase
We set out to produce chitosan hydrogels that use chitin as a source material. Originally, this N-acetylglucosamine oligosaccharide (chitin) is extracted chemically from crustacean shells, which uses a lot of chemicals and produces chitin oligosaccharides of unspecified length. To avoid the usage of chemicals, one aim of this project was to produce chitin in E. coli by insertion of a chitin synthase (CHS) in E. coli, using the BioBrick system.
The CHS can produce chitin in E. coli in an enviromentally friendly way. The enzyme that was employed in this project is the CHS NodC from the bacteria Rhizobium leguminosarum. NodC is an N-acetylglucosaminyl transferase which catalyzes the formation of chitin tetramers and pentamers using activated N-acetylglucosamine monomers. In addition, NodC reliably produces short oligosaccharides of certain lengths which can be processed in vitro further.
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Chitinase
The chitinase is an enzyme, whose ability to break down glycosidic bonds in chitin, brings more variability into the molecules. Since not just the grade and pattern of deacetylation, but also the amount of connected chitin monomers influences the entire molecule´s behavior, there is a great limitation of the properties and the bioactivity of the products. Its possible implementation in the project shows the future prospects of how chitins and chitosans with all kind of properties can be produced in E.coli.
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Chitin Deacetylase
Chitosan is a polymeric product of deacetylated chitin, which exists in a wide variety of patterns differing in their degree of deacetylation. Our goal is to design chitosan oligomers with a specific pattern of deacetylation. It can then be used for the production of our hydrogels.
We implemented chitin deacetylases originating from the organisms Sinorhizobium meliloti (NodB) and Vibrio cholerae (COD) into our E. coli cells. These enzymes deacetylate chitin individually. NodB targets the first position of the non-reducing end, while COD works similarly on the second unit. By regulating this pattern using an orthogonal expression system, designer chitosan can be adjusted to the respective task. Our method allows the expression of each enzyme separately creating a defined deacetylation pattern.
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Hydrogels
Hydrogels are three-dimensional networks made out of synthetic or natural polymers containing a high water content, therefore they receive an increasing attention in various fields. We were focused on not using any expensive or toxic compounds in combination with chitosan to manufacture a hydrogel. It could be formed at any shape with the perfect alignment to the surrounding tissue. The aim was to produce such a hydrogel with basic lab equipment and modify it to detect pathogenic bacteria visually in wounds. To evaluate an ideal hydrogel, various compounds like agarose, agar or alginate were tested. During this work various promising hydrogels were produced.
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Chemistry
Chitosan can be modified at the amino group with succinyl anhydride and a variable peptide with a fluorogenic substrate to form a reliable structure to detect proteases. In this study, we reproduce the findings of the paper “Enzyme-Sensing Chitosan Hydrogels” by Mir Morteza Sadat Ebrahimi and Holger Schönherr from the university of Siegen [1] and use the fluorogenic substrate alanyl-alanyl-phenylalanine-7-amido-4-methylcoumarin (Ala-Ala-Phe-AMC) to detect α-chymotrypsin. This protease is secreted by Staphylococcus aureus or Pseudomonas aeruginosa that are examples of pathogenic bacteria that can infect wounds.
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