Team:Nevada/RD29A

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== Promoters ==
== Promoters ==
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https://static.igem.org/mediawiki/2010/9/98/Finished_final.png '''RD29A Promoter + Strong Plant Kozak (RBS) + RFP''' [[Team:Nevada/registry submissions]]
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'''RD29A Promoter + Strong Plant Kozak (RBS) + RFP''' [[Team:Nevada/registry submissions]]
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<p>The 2010 Nevada iGEM team has three goals for this year’s competition.  First, 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 competitions. This 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>In our project, we designed the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter and the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter + Strong Plant Kozak (RBS) + RFP. These both came from the synthetic design that contained the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter + Strong Plant Kozak (RBS) + RFP in the pMA vector.
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The <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> promoter was isolated by the use of the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> designed primers in PCR. This was blunt end Topo cloned, digested with EcoR 1 and Pst 1 sites, and was then ligated to the pSB1C3 vector.
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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 interest. We 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 standards. Lastly, 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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The design of the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter + Strong Plant Kozak (RBS) + RFP involved the ligation to the pSB1C3 vector using the EcoR 1 and Pst 1 sites. This composite part contains both the promoter and the reporter gene. This functions when the promoter is activated during environmental stress, which would then allow the expression of red fluorescence in plants that can be used as a warning signal to farmers that their plants are under stress.</p>
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<br>'''References'''
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'''Cong L, Zheng H, Zhang Y, Chai T.''' Arabidopsis DREB1A confers high salinity tolerance and regulates the expression of GA dioxygenases in Tobacco. Plant Science [serial online]. February 2008;174(2):156-164. Available from: Academic Search Premier, Ipswich, MA. Accessed October 24, 2010.
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'''Babak B, Akira K, Fevziye C, Mie K, Kazuko Y, Kazuo W.''' Arabidopsis rd29A::DREB1A enhances freezing tolerance in transgenic potato. Plant Cell Reports [serial online]. August 26, 2007;26(8):1275-1282. Available from: Academic Search Premier, Ipswich, MA. Accessed October 25, 2010.
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'''We would like to thank the following sponsors for their support in helping us make this project possible.'''
 
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Much thanks to the [http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology] and the [http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources] for their encouragement and support. Thank you [http://www.unr.edu/inbre/ Nevada INBRE] for over $6,000 in support for supplies and registration costs.
 
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Thank you to [https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada] for supporting our fund raising efforts.
 
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Thank you to [http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.] for free enzyme donations.
 
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Thank you to [http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.] for a discount on our Vector NTI program.
 
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Latest revision as of 21:42, 27 October 2010


Promoters

Finished_final.png RD29A Promoter + Strong Plant Kozak (RBS) + RFP Team:Nevada/registry submissions

In our project, we designed the rd29A Promoter and the rd29A Promoter + Strong Plant Kozak (RBS) + RFP. These both came from the synthetic design that contained the rd29A Promoter + Strong Plant Kozak (RBS) + RFP in the pMA vector. The rd29A promoter was isolated by the use of the rd29A designed primers in PCR. This was blunt end Topo cloned, digested with EcoR 1 and Pst 1 sites, and was then ligated to the pSB1C3 vector. The design of the rd29A Promoter + Strong Plant Kozak (RBS) + RFP involved the ligation to the pSB1C3 vector using the EcoR 1 and Pst 1 sites. This composite part contains both the promoter and the reporter gene. This functions when the promoter is activated during environmental stress, which would then allow the expression of red fluorescence in plants that can be used as a warning signal to farmers that their plants are under stress.



References
Cong L, Zheng H, Zhang Y, Chai T. Arabidopsis DREB1A confers high salinity tolerance and regulates the expression of GA dioxygenases in Tobacco. Plant Science [serial online]. February 2008;174(2):156-164. Available from: Academic Search Premier, Ipswich, MA. Accessed October 24, 2010.
Babak B, Akira K, Fevziye C, Mie K, Kazuko Y, Kazuo W. Arabidopsis rd29A::DREB1A enhances freezing tolerance in transgenic potato. Plant Cell Reports [serial online]. August 26, 2007;26(8):1275-1282. Available from: Academic Search Premier, Ipswich, MA. Accessed October 25, 2010.


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