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Team:Hannover/Parts
From 2014.igem.org
<groupparts>iGEM013 Hannover</groupparts>
Concept
Our goal was to engineer a transgenic plant, which is able to bind heavy metals like cadmium, arsenic, zinc and copper. Therefor metal binding proteins were fused (called top four metal binding protein, T4MBP). To avoid cytotoxic effects on the plant caused by the heavy metals we decided to place T4MBP to the cell exterior. With a secretion signal (Expa4) the T4MBP is directed to the extracellular space. The protein is tethered to the cell surface by using a cellulose binding domain (CBD). Plant cells have a cell wall which consists of cellulose. Via the cellulose binding domain the protein is attached to the cell wall.
Expa4 secretion signal, localizes to extracellular space (BBa_K1478000)
Coding region for first 20 aminoacids of plant protein Exansin4. Secretion signal for plants, when fused to a protein directs the protein to the extracellular space. Derived from the signal sequence of Expansin4 protein from Arabidopsis thaliana (NP_181500). The sequence was optimized for Escherichia coli and Arabidopsis thaliana codon usage. Analyzes showed that the part works.
Analyzes
Bioinformatic analyses based on the raw protein sequence (20 aa) predicts the secretion of the protein with 50 % probability (fig. 1).
For further analyses a GFP-fusion protein was designed. Therefor Expa4 was cloned in frame with GFP and the cellulose binding domain. This coding sequence was cloned into a vector backbone and was used for plant transformation. The GFP signal was detected via confocal microscopy in plant cells. In large eukaryotic cells it’s possible to analyze the subcellular localization with fusion proteins [1]. A prediction via bioinformatics showed nearly 100 % probability for the secretion of the fusion protein (fig. 2).
