Introduction

Chitin is a molecule found in fungal cell walls [1]. Many plants possess enzymes, so-called chitinases, which are able to break down chitin and thus help along with its digestion. These enzymes play a role in defense mechanisms of plants, in case of fungal infections [2]. Even in human tissues chitinases appear, where they defend us against parasites [3]. Chitinases break down the glycosidic bonds between chitin monomer units, and are classified as hydrolases.
In our project, we decided to focus mainly on chitosan pentamers, synthesized and deacetylated by NodC and NodB respectively. However, chitosan's bioactivity and properties are also defined by its polymer length. For this reason, we want to give a first look at how our project could be extended.
The enzyme we use is the ChiA1 from Bacillus circulans [4]. In the strain WL-12, the gene chiA encodes, together with chiB, chiC and chiD, an entire chitinase-system which primarily degrades chitin. This system is made up by at least six different chitinases [5].
Since ChiA1 has been successfully expressed together with our chitin deacetylase NodB [6], we originally decided on this specific enzyme. Its enzymatic activity has also previously been tested on chitin pentamers. ChiA1 breaks down these chitin pentamers in two dimers and one GlcNAc unit [6]. Its implementation would then give us a bigger variety of chitosan molecules.
The N-terminal domain in the ChiA1 is responsible for its catalytic activity. The C-terminal domain plays an important role in the hydrolysis of chitin [6][7].

Methods

We ordered the chiA gene via IDT sequencing. First, we inserted this gene into the pUPD vector using a GoldenBraid assembly as this is a simple and fast cloning method [9]. For cloning the chiA gene into the pSB1C3 vector, we used the BioBrick system [10].
After cutting the pUPD vector, which contains the chiA gene and the pSB1C3 vector, with the restriction enzymes Xba1 and Pst1, dephosphorylating the backbone and sbsequent ligation, we inserted the chiA gene succesfully into the pSB1C3 vector with an Anderson promotor with defined cleavage sites (BBa_K2380025).
We used the same protocols inserting the chiA gene succesfully in the pSB1C3 vector with an inducible promotor system. We altered the restriction enzymes to Nhe1 and Pst1 for the pUPD vector with the chiA gene in order to exchange the Anderson promotor which is located on the pUPD vector. We have cut the pSB1C3 vector containing an AraC promotor system (BBa_K808000) with the restriction enzymes Spe1 and Pst1, dephosphorylated it and succesfully inserted the chiA gene via subsequent ligation. As Nhe1 and Spe1 are complementary the finalized construct does contain the BioBrick retriction sites.
Both the pSB1C3 vectors, containing the chiA gene, have the RBS BBa_K2380024. After that, we transformed both plamids into Top10 cells and BL21 cells (x). We verified the validity via eurofins tube sequencing (x), using a Mini-Prep-Kit first for DNA preparation.
We induced the BL21 cells, containing the pSB1C3 vector with the AraC promotor system, with arabinose to start expression. In order to validate the successful expression, we performed a SDS-Page.

References

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