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Revision as of 11:28, 15 October 2017
ChiTUcare
Chitinase A1
Abstract
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.
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 [1]. For cloning the chiA gene into the pSB1C3 vector, we used the BioBrick system [2].
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 1g/ml arabinose to start expression. In order to validate the successful expression, we performed a SDS-Page.
References
| [1] | GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology
Alejandro Sarrion-Perdigones, Marta Vazquez-Vilar, Jorge Palací, Bas Castelijns, Javier Forment, Peio Ziarsolo, José
Blanca, Antonio Granell, Diego Orzaez
Plant Physiology Jul 2013, 162 (3) 1618-1631 DOI:10.1104/pp.113.217661 |
