Background / Bioinformatics - building a multiple metal binding protein

At the beginning, we started an extensive research to find suitable sequence parts for our own metal binding protein (T4MBP) and the cellulose binding domain (CBD). The consideration of the sequence origin is an important aspect at the beginning of very project for biosafety reasons. Therefore, we studied reports in large scale to get impressions about studies, experiments and insights of these genes. With these information, we were able to estimate the properties of metal binding for these proteins.

We found four metal binding amino acid sequences and a cellulose binding domains. Our overall goal was to obtain a transgenic plant expressing the goi´s. In a next step, the codon usage was adapted by reverse translation of the corresponding amino acid sequences into DNA sequences under consideration of the codon usage (Escherichia coli and Arabidopsis thaliana). Simultaneously, we eliminated unwanted sites via silent mutation. Before sequence assembling, we inserted one Serin-Glycin Linker between each part and the CBD. To suppress RNAi effects, the codon usage was altered by silent mutantion.

To predict the starting point of translation, we inserted our combined insert into the vector and used bioinformatics tool. Caused by the regimentations of iGEM we removed all forbidden restrictionsites. As final step, we integrated a highly efficient 5´UTR to enhance expression.