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From 2014.igem.org

Concept / Old routes – new ideas

Since 2007, 17 teams from 8 different countries have already chosen the problem of heavy metals in our environment as their topic. A total of 13 medals have been awarded in this subject area. This shows how relevant and highly topical the subject is. While other teams have concentrated more on detection and quantification, we want to go one step further with our concept of plant-based water and soil decontamination. Here you find more Background about heavy metals...

With our project we combine cleaning nature by using nature itself: We want to equip plants with a protein which binds four different heavy metals at the same time and hence brings about a significant reduction in the heavy metal concentration of grounds and seas. We thus hope to achieve more extensive binding of hazardous heavy metals than that achieved by conventional methods.

1. step: Transformation of Plants	2. step: Secretion and Immobilising	3. step: Water and soil decontamination

Our path to finding a solution

We created a protein for binding heavy metals that is intended to be expressed and produced in plants. This protein, called Top4-Metal-Binding-Protein (T4MBP), contains 4 different domains of methallothioneins. Each domain originates from a different organism and is specific to a different heavy metal: Copper, Arsenic, Zinc and Cadmium. To avoid cytotoxic effects on the plant caused by the heavy metals, we decided to put T4MBP out of the cell. With a secretion signal (Expa4) the T4MBP is directed to the extracellular space. Via a fused cellulose binding domain (CBD) at the C-term, the protein is attached to the cell wall. We completed this construct with an optimized 5'UTR. The entire construct was synthesized and cloned into the target vector: The binary vector pORE_E3 in which we exchanged the promoter with a 2x35S promoter. This pORE_E3_2x35S_T4MBP_CDB vector were used to transform our plants. Here you find a nice picture showing our construct...

We decided to demonstrate the principle initially in the model organisms Arabidopsis thaliana (A. thaliana) and Nicotiana tabacum (N. tabacum) with the aid of a transformation by Rhizobium radiobacter (formerly Agrobacterium tumefaciens). For what reason...?

Project overview

Steps	Subproject	How?
1.	Constuction of our T4MBP sequenz	Selection, research and bioinformatics How...?
1.1	T4MBP Plant	Stabile transformation of A. thaliana For what reason...?
1.2 A	Heterologous expression of our T4MBP	Using E. coli as an expression system For what reason...?
1.2 B	Heterologous expression of our T4MBP	Using N. tabacum as an transient expression system
1.2.1	Quantitative Analysis of our T4MBP	ICP-OES analysis What´s that...?
1.3	GFP: Locate the CBD precisely within a plant cell	CBD fused with GFP, insert it transiently via leaf-infiltration and take a look using a confocal microscope
2.	Plant vector RFC[21]	We want to provide iGEM a new Plant vector with BioBrick RFC[21]MCS and 2x35S promotor. For what reason...?