Team:Groningen/Project

From 2009.igem.org

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==Teams with similar projects==
 
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:→ [http://2009.igem.org/Team:UQ-Australia UQ Australia]
 
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Water contamination is a key environmental issue for many countries around the world, both developed and developing.  In Queensland, Australia we have a particular problem with Mercury (Hg<sup>2+</sup>) contamination of water supplies around the major mining town of Mt Isa. After searching through the iGEM projects from previous years,  the arsenic detection system inspired us.  As the UQ 09' team we wish to take this idea one step further and completely remove Mercury from water systems.
 
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To do this we will be utilizing a strain of ''Escherichia coli'', and the already established mercury uptake, reduction and efflux system and making a few modifications. One of our aims is to couple the detection of Mercury to the expression of a native bacterial protein, Antigen 43 (AG43).  This protein, when expressed, causes the bacteria to stick to one another. As the bacteria aggregate in clumps, they will fall to the bottom of the sample.  Our idea is for the bacteria to take up the mercury, activating  Ag43 expression, resulting in aggregation and the Mercury-filled bacteria will fall to the bottom leaving clean water.
 
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There are a number of parts that we hope to add to the registry. The first is Ag43 as a protein coding sequence and the MerR promoter sequence.  We will also add the completed mercury uptake and aggregation system as an operon.
 
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:→ [http://2009.igem.org/Team:Newcastle Newcastle]
 
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The aim of our project is to genetically engineer ''Bacillus subtilis'' to be able to detect and sense cadmium which has been taken up from the soil environment and sequester them into a metallothionein. This metallothionein will then become incorporated into a Bacillus spore; the resilience of which means that the cadmium ions can become isolated from the environment (and made bio-unavailable) for many years.
 
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This project involves a number of steps, each of which can be considered as sub projects:
 
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# Metal intake
 
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# Metal sensing
 
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# Tuning of ''Bacillus subtilis'' normal stochastic switch
 
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# Metal sequestration by metallothionein
 
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# Second stochastic switch
 
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# Synthesizing a Promoter Library for ''Bacillus subtilis''
 
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==Periodic table==
 
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In the periodic table below you can see for which elements we have identified a transporter, an accumulating protein and/or promotor.
 
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{{periodic_table
 
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|AsInfo=
 
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Transporter: GlpF<br/>
 
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Accumulator: ArsR<br/>
 
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|SbInfo=
 
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Transporter: GlpF<br/>
 
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|CuInfo=
 
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Transporter: {{part|BBa_K190018|HmtA}}<br/>
 
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Accumulator: SmtA<br/>
 
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|ZnInfo=
 
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Transporter: {{part|BBa_K190018|HmtA}}<br/>
 
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Accumulator: SmtA<br/>
 
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|HgInfo=
 
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Transporter: MerT<br/>
 
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Accumulator: SmtA<br/>
 
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|CdInfo=
 
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Accumulator: SmtA<br/>
 
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}}
 
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{{Team:Groningen/Footer}}
{{Team:Groningen/Footer}}

Revision as of 19:00, 21 October 2009

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The Project
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Heavy metal scavengers with a vertical gas drive

Introduction:

Human health and the environment are endangered by heavy metal pollution in water and sediment. To improve purification strategies a metal selective microbacterial cleaning system was designed. The system comprises uptake, sequestering and metal sensitive buoyancy. All subsystems are interchangeable, which makes it suitable for almost any metal cleaning assay. For this project the modular system was focused on arsenic accumulation, using Escherichia coli as a chassis organism. Arsenite and arsenate are imported by GlpF, a aquaglycerol porin from E. coli. Intracellular As(III) and As(V) are sequestered by fMT or ArsR. These proteins were used as the accumulation modules. Since E. coli does not have a buoyancy system, the polycistronic gas vesicle protein gene cluster from Bacillus megaterium, GVP, was used. The arsenic promoter from E. coli, pArsR, is regulated by the negative transcriptional regulator ArsR. GVP, under regulation of pArsR, was used as the metal sensitive buoyancy module.


Results:

All modules were cloned according to the BioBrickTM Standard Assembly 10. The synthetic gene GlpF , was successfully cloned into a synthetic operon, with fMT. The GVP cluster, with a ten times repeat sequence, was successfully cloned downstream of the pArsR promoter. These two subsystems were transformed in E. coli to complete the system. The system and its subparts were tested using several assays. Accumulation was tested by an uptake assay, however, since no reproducible results were obtained, the functionality of the accumulation module could not be determined from these data. Arsenic uptake was examined using a metal sensitivity assay. The E. coli strain overexpressing GlpF showed a decreased final cell density upon induction with As(III), suggesting functional expression of the transporter. The metal sensitive promoter pArsR was tested using a fluorescence assay. This showed a 2.3 fold increased activity upon induction with 100 µM NaAsO2. Buoyancy was tested by a sedimentation assay. Enhanced buoyancy was shown for the buoyancy module and the complete system, though no difference of the buoyancy phenotype could be observed upon addition of the accumulation module. Cells cultivated in aerobic conditions showed improved buoyancy compared to cells cultivated in semi-aerobic conditions. Expression of gas vescicles was shown by electron microscopy. An interactive computer model was made for the whole system, with which the modules were further characterized. With the model, import rates of As(III) at different initial extracellular arsenic concentrations could be determined. Also the influence of different parameters on the accumulation factor, the ratio between bound and unbound arsenic, was calculated. The model also allowed qualitative determination of the regulation of pArsR by various expression levels of ArsR. Furthermore, the volume fraction gas vesicles in the cells needed for buoyancy, for several sizes of the gas vesicles, was computed.

Conclusion:

The metal selective microbacterial cleaning system for arsenic was shown to be buoyant and the buoyancy module and uptake module were shown to work individually. For a better determination of the system an accumulation assay need to be redone. It was shown here that the system has potential as a cleaning system for arsenic. As mentioned earlier this modular system can also be implemented in cleaning of other substances. Literature research showed possible modules for copper, zinc, mercury and even gold. So not only cleaning water and sludge but also mining rare metals could be functionalized using this system.