Team:Nevada/CD2Inducible

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<p>The 2010 Nevada iGEM team has three goals for this year’s competitionFirst, we are going to test the validity of utilizing Nicotiana tabacum protoplasts (NT cells), plant cells without the cell wall, as a model for the expression of higher plant genes for future iGEM competitionsThis system is useful in the respect that the time it takes to obtain transgenic lines of cells is greatly reduced compared to the time to obtain transgenic plants. <html>
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<p>Heavy metal contamination is an important environmental issueHeavy metals can contaminate soil and water sources in areas where mining and various industrial proccesses have occurred.  These metals can then be absorbed into plants which are subsequently eaten by various animals.  Heavy metals are usually not excreted readily and are retained within the body of an animal that has consumed a contaminated food sourceCadmium in particular is not excreted readily from the mammals and is known to cause various etiologies stemming from its build-up in organs (Gobe). <html>
<a href="https://static.igem.org/mediawiki/2010/7/79/GFP_tobacco_plant.jpg"><img src="https://static.igem.org/mediawiki/2010/7/79/GFP_tobacco_plant.jpg" class="shadow" style="float:left;width:170px;margin:10px"></a>
<a href="https://static.igem.org/mediawiki/2010/7/79/GFP_tobacco_plant.jpg"><img src="https://static.igem.org/mediawiki/2010/7/79/GFP_tobacco_plant.jpg" class="shadow" style="float:left;width:170px;margin:10px"></a>
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</html>These cells can therefore be utilized as a quick proof-of-concept test model before moving synthetic constructs into plants of interestWe also aim to produce an iGEM-compatible plant-specific plasmid, several stress-inducible plant promoters, reporter genes containing Kozak sequences (ribosome binding sites) and terminators that conform to BioBrick standardsLastly, we hope to measure the induction of these stress promoters in real-time by performing a fluorometry assay in which stress will be applied to NT cells and fluorescent output by a reporter (GFP) will be measured to detail induction in real time.  This method has a distinct advantage over microarrays since microarrays are only one ‘snapshot’ in time.</p>
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</html>In order to develop a mock cadmium-sensing system in plants the promoter for the Cd-transporter gene AtMRP3 (At3g13080) from A. thaliana was transformed into N. tabacum cellsAtMRP3 is utilized by the plant to sequester Cd2+ in the vacuole, which is thought to prevent the cation from interfering with various biological processes (Zientara et al.).  Besides being highly induced by cadmium, AtMRP3 has also shown similar induction patterns when plants were subjected to arsenic or lead, thusly making it a useful sensor for various heavy metal soil contaminants.
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AtMRP3 will be the first plant-compatible heavy metal promoter available to the iGEM registry.  This promoter could be coupled with a myriad of reporters to indicate whether or not plants are experiencing any type of stress due to the presence of cadmium or other heavy metals.</p>

Revision as of 18:39, 14 October 2010



Promoters

Heavy metal contamination is an important environmental issue. Heavy metals can contaminate soil and water sources in areas where mining and various industrial proccesses have occurred. These metals can then be absorbed into plants which are subsequently eaten by various animals. Heavy metals are usually not excreted readily and are retained within the body of an animal that has consumed a contaminated food source. Cadmium in particular is not excreted readily from the mammals and is known to cause various etiologies stemming from its build-up in organs (Gobe). In order to develop a mock cadmium-sensing system in plants the promoter for the Cd-transporter gene AtMRP3 (At3g13080) from A. thaliana was transformed into N. tabacum cells. AtMRP3 is utilized by the plant to sequester Cd2+ in the vacuole, which is thought to prevent the cation from interfering with various biological processes (Zientara et al.). Besides being highly induced by cadmium, AtMRP3 has also shown similar induction patterns when plants were subjected to arsenic or lead, thusly making it a useful sensor for various heavy metal soil contaminants. AtMRP3 will be the first plant-compatible heavy metal promoter available to the iGEM registry. This promoter could be coupled with a myriad of reporters to indicate whether or not plants are experiencing any type of stress due to the presence of cadmium or other heavy metals.






We would like to thank the following sponsors for their support in helping us make this project possible. Much thanks to the Departments of Biochemistry and Biotechnology and the College of Agriculture, Biotechnology and Natural Resources for their encouragement and support. Thank you Nevada INBRE for over $6,000 in support for supplies and registration costs. Thank you to Associated Students of the Univeristy of Nevada for supporting our fund raising efforts. Thank you to Promega Co. for free enzyme donations. Thank you to Invitrogen Co. for a discount on our Vector NTI program.

Nevada CABNR.jpg NV INBRE Logo.jpg UNR ASUN logo.jpg Promega logo.jpg Invitrogen logo.jpeg