Team:Hannover/Project

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<h1>Project description</h1>
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<h1>Concept / Old routes – new ideas</h1>
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<h2>Heavy metals – topical and ubiquitous</h2>
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<p class="text">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.<a href="https://2014.igem.org/Team:Hannover/Background_Heavy_metals"> Here you find more background about heavy metals...</a><br><br>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 a significant reduction in the heavy metal concentration of grounds and seas about. We thus hope to achieve more extensive binding of hazardous heavy metals than that achieved by conventional methods.</p>
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<p class="text">In today’s world, heavy metals are the indispensable basis for the technical progress of our society. They allow us to refine raw materials to improve the infrastructure (copper, zinc), keep us mobile (copper, zinc) and ensure energy is always available from rechargeable batteries (cadmium) and in the form of electricity (copper). The mining industry has grown considerably and the mining of heavy metals has increased dramatically since the Industrial Revolution in the 19th century. This is down to the increase in global demand as well as to new extraction technologies. But what is the real price we have to pay for heavy metals?</p>   
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<p class="text">In nature, heavy metal ions are usually found in complex compounds. The process of mining for gold, lead or copper brings these ions together with large quantities of their poisonous ligands, arsenic, for example, to the surface, where they are temporarily stored on slag heaps. Heavy metals are released by corrosion and the extremely high wear and tear suffered by products containing heavy metals (especially in the electronics and automotive industry). They are also mobilized by weathering processes, and then transported from the slag heaps at the mines into our groundwater. From there they get into our drinking water and via agricultural processes they indirectly get into our food chain. Heavy metals can accumulate in the tissues in the body and even very low concentrations can severely harm our health by causing organ failure, infertility or neuronal degeneration. The heavy metal load is a global problem.  Although the heavy metal load in newly industrialized countries such as China or Bangladesh is much higher than in Germany, there are many possible applications for our project at home as well. Brake wear and tire abrasion release large quantities of zinc and copper onto German roads every day. The rain washes these heavy metals onto the grass verges and water retention basins adjoining the ‘autobahns’ or they enter the surrounding lakes and rivers via the municipal sewerage systems.</p>
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<h2>Old routes – new ideas</h2>
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<p class="text">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. We want to equip plants with a protein which binds several heavy metals at the same time and hence brings about a significant reduction in the heavy metal concentration. We thus hope to achieve more extensive binding of hazardous heavy metals than that achieved by conventional methods.</p>
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<h2>Our path to finding a solution</h2>
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<p class="text">Our goal is to produce a Top4 Metal Binding Protein (T4-MBP) which attaches itself to the cellulose of the plants and binds four heavy metals of global relevance. We intend to use naturally occurring metallothioneins - proteins whose specific amino acid sequences alone make them able to form complexes with heavy metals. We have decided on the following heavy metals or domains and combined them to our first synthesis construct on the basis of the cDNA sequences:</p>
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<p class="text">1. step: Transformation of plants</p>
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<p class="text">The aim is to be able to use the protein in terrestrial (A. thaliana) as well as aquatic plants (Wolffia). Since no-one has had much experience with the transformation of Wolffia, we decided to demonstrate the principle initially in the model organisms A. thaliana and N. benthamiana. Our genetic construct is ultimately to be brought into the target organism with the aid of a transformation by Rhizobium radiobacter (formerly Agrobacterium tumefaciens).</p>
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<p class="text">2. step: Secretion and immobilizing</p>
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<p class="text">3. step: Water and soil decontamination</p>
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<h1>Plant Against: Our path finding a solution</h1>
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<p class="text">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 was used to transform our plants.
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<p class="text">We decided to demonstrate the principle initially in the model organisms <i>Arabidopsis thaliana</i> (<i>A. thaliana</i>) and <i>Nicotiana tabacum</i> (<i>N. tabacum</i>) with the aid of a transformation by <i>Rhizobium radiobacter</i> (formerly <i>Agrobacterium tumefaciens</i>).<a href="https://2014.igem.org/Team:Hannover/Background_Arabidopsis"> For what reason...?</a></p>
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<h1>Project overview</h1>
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<tr><td><h4>Steps</h4></td><td><h4>Subproject</h4></td><td><h4>How?</h4></td></tr>
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<tr><td>1.</td><td>Construction of our T4MBP sequence</td><td>Selection, research and bioinformatics</i><a href="https://2014.igem.org/Team:Hannover/Background_bioinformatics"> How...?</a></td></tr>
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<tr><td>1.1</td><td>T4MBP Plant</td><td>Solid transformation of <i>A. thaliana</i><a href="https://2014.igem.org/Team:Hannover/Background_Arabidopsis"> For what reason...?</a></td></tr>
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<tr><td>1.2 A</td><td>Heterologous expression of our T4MBP</td><td>Using <i>E. coli</i> as an expression system<a href="https://2014.igem.org/Team:Hannover/Background_pASK"> For what reason...?</a></td></tr>
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<tr><td>1.2 B</td><td>Heterologous expression of our T4MBP</td><td>Using <i>N. tabacum</i> as an transient expression system</td></tr>
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<tr><td>1.2.1</td><td>Quantitative analysis of our T4MBP</td><td>ICP-OES analysis <a href="https://2014.igem.org/Team:Hannover/Background_ICP_OES"> What´s that...?</a></td>
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<tr><td>1.3</td><td>GFP: localization of the CBD within a plant cell</td><td>CBD fused with GFP, transiently inserted via leaf-infiltration and taking a look using a confocal microscope</td></tr>
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<tr><td>2.</td><td>Plant vector RFC[21]</td><td>We want to provide iGEM a new plant vector with BioBrick RFC[21]MCS and 2x35S promotor. <a href="Background_Plant_Vector">For what reason...?</a></td></tr>
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Latest revision as of 19:38, 17 October 2014

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 a significant reduction in the heavy metal concentration of grounds and seas about. 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 immobilizing

3. step: Water and soil decontamination

Plant Against: Our path 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 was used to transform our plants.

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.Construction of our T4MBP sequenceSelection, research and bioinformatics How...?
1.1T4MBP PlantSolid transformation of A. thaliana For what reason...?
1.2 AHeterologous expression of our T4MBPUsing E. coli as an expression system For what reason...?
1.2 BHeterologous expression of our T4MBPUsing N. tabacum as an transient expression system
1.2.1Quantitative analysis of our T4MBPICP-OES analysis What´s that...?
1.3GFP: localization of the CBD within a plant cellCBD fused with GFP, transiently inserted via leaf-infiltration and taking 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...?





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