http://2010.igem.org/wiki/index.php?title=Special:Contributions/Mpolasko&feed=atom&limit=50&target=Mpolasko&year=&month=2010.igem.org - User contributions [en]2024-03-28T21:51:51ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:NevadaTeam:Nevada2010-10-27T22:40:46Z<p>Mpolasko: </p>
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<div>{{nevadamain}}<br />
== Abstract ==<br />
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<html><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/8/8b/IGEM_Team_2010.jpg/400px-IGEM_Team_2010.jpg" class="shadow" style="float:right"><br />
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<p>Our long term goals are to develop plant biosensors that reliably measure changes in the local environment. In the process of developing biosensors, the''' 2010 Nevada iGEM Team''' need to develop fundamental promoters, reporters, and plasmids for plants. Therefore, 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 (NT cells) as a model for the expression of higher plant genes for future iGEM competitions. NT cells are a faster, cheaper, and safer model than traditional plant transformation. 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><br />
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<html><a href="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png"><img src="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
</html><br />
<p style="text-align:right;"><span style="font-size:10px;">Fluorescent Plant Image Taken From: http://www.edinformatics.com/inventions_inventors/genetic_engineering.htm</span></p><br />
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'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
Thank you to the <span style="font-weight:bold;">[http://sparks22.adventistchurchconnect.org/ Sparks Seventh-day Adventist Church]</span> for the $1,000 donation in response to the sponsorship letters.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:NevadaTeam:Nevada2010-10-27T22:25:37Z<p>Mpolasko: </p>
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<div><div style="background-color:#DFF7DC;width:970px;"><br />
{{nevadamain}}<br />
== Abstract ==<br />
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<html><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/8/8b/IGEM_Team_2010.jpg/400px-IGEM_Team_2010.jpg" class="shadow" style="float:right"><br />
</html><br />
<br />
<br><br />
<p>Our long term goals are to develop plant biosensors that reliably measure changes in the local environment. In the process of developing biosensors, the''' 2010 Nevada iGEM Team''' need to develop fundamental promoters, reporters, and plasmids for plants. Therefore, 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 (NT cells) as a model for the expression of higher plant genes for future iGEM competitions. NT cells are a faster, cheaper, and safer model than traditional plant transformation. 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><br />
<br><br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png"><img src="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
</html><br />
<p style="text-align:right;"><span style="font-size:10px;">Fluorescent Plant Image Taken From: http://www.edinformatics.com/inventions_inventors/genetic_engineering.htm</span></p><br />
<br />
----<br />
<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
Thank you to the <span style="font-weight:bold;">[http://sparks22.adventistchurchconnect.org/ Sparks Seventh-day Adventist Church]</span> for the $1,000 donation in response to the sponsorship letters.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}<br />
</div></div>Mpolaskohttp://2010.igem.org/Team:NevadaTeam:Nevada2010-10-27T22:19:14Z<p>Mpolasko: </p>
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<div><div style="background-color:#B5EAAA;width:970px;"><br />
{{nevadamain}}<br />
== Abstract ==<br />
<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/8/8b/IGEM_Team_2010.jpg/400px-IGEM_Team_2010.jpg" class="shadow" style="float:right"><br />
</html><br />
<br />
<br><br />
<p>Our long term goals are to develop plant biosensors that reliably measure changes in the local environment. In the process of developing biosensors, the''' 2010 Nevada iGEM Team''' need to develop fundamental promoters, reporters, and plasmids for plants. Therefore, 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 (NT cells) as a model for the expression of higher plant genes for future iGEM competitions. NT cells are a faster, cheaper, and safer model than traditional plant transformation. 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><br />
<br><br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png"><img src="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
</html><br />
<p style="text-align:right;"><span style="font-size:10px;">Fluorescent Plant Image Taken From: http://www.edinformatics.com/inventions_inventors/genetic_engineering.htm</span></p><br />
<br />
----<br />
<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
Thank you to the <span style="font-weight:bold;">[http://sparks22.adventistchurchconnect.org/ Sparks Seventh-day Adventist Church]</span> for the $1,000 donation in response to the sponsorship letters.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}<br />
</div></div>Mpolaskohttp://2010.igem.org/Team:Nevada/TeamTeam:Nevada/Team2010-10-27T04:44:15Z<p>Mpolasko: </p>
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<div>{{Nevada_css}}<br />
[[Image:Picture Team.png|border|left|950px]]<br />
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<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
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<p style="text-align:left;"><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&spades;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&hearts;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&clubs;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&diams;</span>&nbsp;<span style="font-family:Arial;font-size:16pt;text-decoration:bold;color:#15317E;background-color:ffffff;">Meet the Nevada Team</span>&nbsp;<span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&diams;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&clubs;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&hearts;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&spades;</span>&nbsp;&nbsp;&nbsp;&nbsp;</p><br />
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<a href="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG" class="shadow" style="float:left;margin:0px;width:600px"></a><br />
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<p style="text-align:center;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Advisors</span><br />
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[[Image:Christie.jpg|center|thumb|alt=alt text|<p style="text-align:center;"><span style="font-family:Times;font-size:12pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Dr. Christie Howard</span></p>]]<br />
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[[Image:Shintani.jpg|center|thumb|alt=alt text|<p style="text-align:center;"><span style="font-family:Times;font-size:12pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Dr. David Shintani</span></p>]]<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Undergraduates</span><br />
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<gallery widths=170px heights=170px perrow=4><br />
Image:Hilary UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span></p><br />
Image:mpolasko2.JPG|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span></p><br />
Image:Randy.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span></p><br />
Image:Copley.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span></p><br />
Image:Bryson UNR 2.png| <p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span></p><br />
Image:Gladwill.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span></p><br />
Image:Image-Elaine.jpeg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span></p><br />
Image:Sam final UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span></p><br />
&nbsp;<br />
Image:Nick UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span></p><br />
Image:Hileary.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span></p><br />
</gallery><br />
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<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:normal;text-decoration:bold;color:#15317E;background-color:ffffff;">What We Did</span><br />
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{| border="0" cellpadding="0" cellspacing="20"<br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Team President</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">NT Cell Transformations</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter + Plant Kozak + RFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + EYFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + mCherry</span><br />
*<span style="font-size:12pt;font-weight:bold;">Composite - 35s Promoter + Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;"> DREB1C Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">35S Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Modeling</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">RD29A Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|&nbsp;<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span><br />
|-<br />
|&nbsp;<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|}<br />
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</div><br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/TeamTeam:Nevada/Team2010-10-27T04:34:28Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Picture Team.png|border|left|950px]]<br />
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<p>&nbsp;</p><br />
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<p style="text-align:left;"><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&spades;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&hearts;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&clubs;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&diams;</span>&nbsp;<span style="font-family:Arial;font-size:16pt;text-decoration:bold;color:#15317E;background-color:ffffff;">Meet the Nevada Team</span>&nbsp;<span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&diams;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&clubs;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&hearts;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&spades;</span>&nbsp;&nbsp;&nbsp;&nbsp;[[Media:mpolasko_pptdemo.ppt]]</p><br />
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<a href="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG" class="shadow" style="float:left;margin:0px;width:600px"></a><br />
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<p style="text-align:center;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Advisors</span><br />
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----<br />
<br />
[[Image:Christie.jpg|center|thumb|alt=alt text|<p style="text-align:center;"><span style="font-family:Times;font-size:12pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Dr. Christie Howard</span></p>]]<br />
<br />
[[Image:Shintani.jpg|center|thumb|alt=alt text|<p style="text-align:center;"><span style="font-family:Times;font-size:12pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Dr. David Shintani</span></p>]]<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Undergraduates</span><br />
</p><br />
----<br />
<gallery widths=170px heights=170px perrow=4><br />
Image:Hilary UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span></p><br />
Image:mpolasko2.JPG|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span></p><br />
Image:Randy.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span></p><br />
Image:Copley.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span></p><br />
Image:Bryson UNR 2.png| <p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span></p><br />
Image:Gladwill.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span></p><br />
Image:Image-Elaine.jpeg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span></p><br />
Image:Sam final UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span></p><br />
&nbsp;<br />
Image:Nick UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span></p><br />
Image:Hileary.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span></p><br />
</gallery><br />
<br />
<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:normal;text-decoration:bold;color:#15317E;background-color:ffffff;">What We Did</span><br />
</p><br />
----<br />
<br />
<div style="padding:10px;clear:both"><br />
{| border="0" cellpadding="0" cellspacing="20"<br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Team President</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">NT Cell Transformations</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter + Plant Kozak + RFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + EYFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + mCherry</span><br />
*<span style="font-size:12pt;font-weight:bold;">Composite - 35s Promoter + Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;"> DREB1C Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">35S Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Modeling</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">RD29A Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|&nbsp;<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span><br />
|-<br />
|&nbsp;<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|}<br />
----<br />
<br />
</div><br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/File:Mpolasko_pptdemo.pptFile:Mpolasko pptdemo.ppt2010-10-27T04:33:07Z<p>Mpolasko: </p>
<hr />
<div></div>Mpolaskohttp://2010.igem.org/Team:Nevada/CompositeTeam:Nevada/Composite2010-10-27T04:11:51Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Composite UNR.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
== Composite Parts ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*RD29A Promoter + Strong Plant Kozak (RBS) + RFP [[Team:Nevada/registry submissions]]<br />
*35S Promoter + (Strong Plant RBS + GFP) From K414001 [[Team:Nevada/registry submissions]]<br />
----<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">rd29A – RFP</span>: This composite is designed so that transformed plants will yield red fluorescent protein under cold, drought, or high soil salinity conditions. We were successful in developing cold-responsive red fluorescent NT cells in time for the jamboree. Please check out the <html><a href="https://2010.igem.org/Team:Nevada/Results"><span style="color:#1569C7;font-weight:bold;">Transformation Results</span></a></html>.</p><br />
For more information on rd29A, click <html><a href="https://2010.igem.org/Team:Nevada/RD29A"><span style="color:#1569C7;font-weight:bold;">here</span></a></html>.<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">35S – GFP</span>: This composite is designed so that transformed plants will have constitutive expression of green fluorescent protein. Having this constant expression provides the basis to measure any other inducible fluorescent proteins’ fluorescence relative to an internal control. </p><br />
For more information on 35S, click <html><a href="https://2010.igem.org/Team:Nevada/35S"><span style="color:#1569C7;font-weight:bold;">here</span></a></html>.<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">DREB1C - EYFP</span>: Although not developed in time for this year's iGEM competition. We had planned on developing a composite part that would respond specifically to cold response. This would provide a specific signal and not be as receptive to various stresses like RD29A. Under cold stress, this composite would express enhanced yellow fluorescent protein.</p> <br />
For more information on DREB1C, click <html><a href="https://2010.igem.org/Team:Nevada/DREB1C"><span style="color:#1569C7;font-weight:bold;">here</span></a></html>. <br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
<br />
<br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/TeamTeam:Nevada/Team2010-10-27T04:10:39Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Picture Team.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
<p style="text-align:left;"><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&spades;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&hearts;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&clubs;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&diams;</span>&nbsp;<span style="font-family:Arial;font-size:16pt;text-decoration:bold;color:#15317E;background-color:ffffff;">Meet the Nevada Team</span>&nbsp;<span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&diams;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&clubs;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&hearts;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&spades;</span><br />
</p><br />
----<br />
<div style="padding: 10px 0 10px 10px;"><br />
<br />
<html><br />
<a href="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG" class="shadow" style="float:left;margin:0px;width:600px"></a><br />
</html><br />
<br />
<p style="text-align:center;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Advisors</span><br />
</p><br />
----<br />
<br />
[[Image:Christie.jpg|center|thumb|alt=alt text|<p style="text-align:center;"><span style="font-family:Times;font-size:12pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Dr. Christie Howard</span></p>]]<br />
<br />
[[Image:Shintani.jpg|center|thumb|alt=alt text|<p style="text-align:center;"><span style="font-family:Times;font-size:12pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Dr. David Shintani</span></p>]]<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Undergraduates</span><br />
</p><br />
----<br />
<gallery widths=170px heights=170px perrow=4><br />
Image:Hilary UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span></p><br />
Image:mpolasko2.JPG|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span></p><br />
Image:Randy.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span></p><br />
Image:Copley.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span></p><br />
Image:Bryson UNR 2.png| <p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span></p><br />
Image:Gladwill.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span></p><br />
Image:Image-Elaine.jpeg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span></p><br />
Image:Sam final UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span></p><br />
&nbsp;<br />
Image:Nick UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span></p><br />
Image:Hileary.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span></p><br />
</gallery><br />
<br />
<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:normal;text-decoration:bold;color:#15317E;background-color:ffffff;">What We Did</span><br />
</p><br />
----<br />
<br />
<div style="padding:10px;clear:both"><br />
{| border="0" cellpadding="0" cellspacing="20"<br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Team President</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">NT Cell Transformations</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter + Plant Kozak + RFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + EYFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + mCherry</span><br />
*<span style="font-size:12pt;font-weight:bold;">Composite - 35s Promoter + Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;"> DREB1C Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">35S Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Modeling</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">RD29A Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|&nbsp;<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span><br />
|-<br />
|&nbsp;<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|}<br />
----<br />
<br />
</div><br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/CompositeTeam:Nevada/Composite2010-10-27T04:05:23Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Composite UNR.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
== Composite Parts ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*RD29A Promoter + Strong Plant Kozak (RBS) + RFP [[Team:Nevada/registry submissions]]<br />
*35S Promoter + (Strong Plant RBS + GFP) From K414001 [[Team:Nevada/registry submissions]]<br />
----<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">rd29A – RFP</span>: This composite is designed so that transformed plants will yield red fluorescent protein under cold, drought, or high soil salinity conditions. We were successful in developing cold-responsive red fluorescent NT cells in time for the jamboree. Please check out the <html><a href="https://2010.igem.org/Team:Nevada/Results"><span style="color:#1569C7;font-weight:bold;">Transformation Results</span></a></html>.</p><br />
For more information on rd29A, click <html><a href="https://2010.igem.org/Team:Nevada/RD29A"><span style="color:#1569C7;font-weight:bold;">here</span></a></html>.<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">35S – GFP</span>: This composite is designed so that transformed plants will have constitutive expression of green fluorescent protein. Having this constant expression provides the basis to measure any other inducible fluorescent proteins’ fluorescence relative to an internal control. </p><br />
For more information on 35S, click <html><a href="https://2010.igem.org/Team:Nevada/35S"><span style="color:#1569C7;font-weight:bold;">here</span></a></html>.<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">DREB1C - EYFP</span>: Although not developed in time for this year's iGEM competition. We had planned on developing a composite part that would respond specifically to cold response. This would provide a specific signal and not be as receptive to various stresses like RD29A. Under cold stress, this composite would express enhanced yellow fluorescent protein. <br />
For more information on DREB1C, click <html><a href="https://2010.igem.org/Team:Nevada/DREB1C"><span style="color:#1569C7;font-weight:bold;">here</span></a></html>. <br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
<br />
<br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/CompositeTeam:Nevada/Composite2010-10-27T03:59:00Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Composite UNR.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
== Composite Parts ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*RD29A Promoter + Strong Plant Kozak (RBS) + RFP [[Team:Nevada/registry submissions]]<br />
*35S Promoter + (Strong Plant RBS + GFP) From K414001 [[Team:Nevada/registry submissions]]<br />
----<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">rd29A – RFP</span>: This composite is designed so that transformed plants will yield red fluorescent protein under cold, drought, or high soil salinity conditions. We were successful in developing cold-responsive red fluorescent NT cells in time for the jamboree. Please check out the <html><a href="https://2010.igem.org/Team:Nevada/Results"><span style="color:#1569C7;font-weight:bold;">Transformation Results</span></a></html>.</p><br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">35S – GFP</span>: This composite is designed so that transformed plants will have constitutive expression of green fluorescent protein. Having this constant expression provides the basis to measure any other inducible fluorescent proteins’ fluorescence relative to an internal control. </p><br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">DREB1C - EYFP</span>: Although not developed in time for this year's iGEM competition. We had planned on developing a composite part that would respond specifically to cold response. This would provide a specific signal and not be as receptive to various stresses like RD29A. Under cold stress, this composite would express enhanced yellow fluorescent protein. <br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
<br />
<br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/CompositeTeam:Nevada/Composite2010-10-27T03:55:36Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Composite UNR.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
== Composite Parts ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*RD29A Promoter + Strong Plant Kozak (RBS) + RFP [[Team:Nevada/registry submissions]]<br />
*35S Promoter + (Strong Plant RBS + GFP) From K414001 [[Team:Nevada/registry submissions]]<br />
----<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">RD29A – RFP</span>: This composite is designed so that transformed plants will yield red fluorescent protein under cold, drought, or high soil salinity conditions. We were successful in developing cold-responsive red fluorescent NT cells in time for the jamboree. Please check out the <html><a href="https://2010.igem.org/Team:Nevada/Results"><span style="color:#1569C7;font-weight:bold;">Transformation Results</span></a></html>.</p><br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">35S – GFP</span>: This composite is designed so that transformed plants will have constitutive expression of green fluorescent protein. Having this constant expression provides the basis to measure any other inducible fluorescent proteins’ fluorescence relative to an internal control. </p><br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">DREB1C - EYFP</span>: Although not developed in time for this year's iGEM competition. We had planned on developing a composite part that would respond specifically to cold response. This would provide a specific signal and not be as receptive to various stresses like RD29A. Under cold stress, this composite would express enhanced yellow fluorescent protein. <br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
<br />
<br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/CompositeTeam:Nevada/Composite2010-10-27T03:48:22Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Composite UNR.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
== Composite Parts ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*RD29A Promoter + Strong Plant Kozak (RBS) + RFP [[Team:Nevada/registry submissions]]<br />
*35S Promoter + (Strong Plant RBS + GFP) From K414001 [[Team:Nevada/registry submissions]]<br />
----<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">RD29A – RFP</span>: This composite is designed so that transformed plants will yield red fluorescent protein under cold, drought, or high soil salinity conditions. We were successful in developing cold-responsive red fluorescent NT cells in time for the jamboree. Please check out the <html><a href="https://2010.igem.org/Team:Nevada/Results"><span style="color:#1569C7;font-weight:bold;">Transformation Results</span></a></html>.</p><br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">35S – GFP</span>: This composite is designed so that transformed plants will have constitutive expression of green fluorescent protein. Having this constant expression provides the basis to measure any other inducible fluorescent proteins’ fluorescence relative to an internal control. </p><br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
<br />
<br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/CompositeTeam:Nevada/Composite2010-10-27T03:46:38Z<p>Mpolasko: /* Composite Parts */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Composite UNR.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
== Composite Parts ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*RD29A Promoter + Strong Plant Kozak (RBS) + RFP [[Team:Nevada/registry submissions]]<br />
*35S Promoter + (Strong Plant RBS + GFP) From K414001 [[Team:Nevada/registry submissions]]<br />
----<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;">RD29A – RFP</span>: This composite is designed so that transformed plants will yield red fluorescent protein under cold, drought, or high soil salinity conditions. We were successful in developing cold-responsive red fluorescent NT cells in time for the jamboree. Please check out the <html><a href="https://2010.igem.org/Team:Nevada/Results"><span style="color:#1569C7;font-weight:bold;">Transformation Results</span></a></html>.</p><br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;">35S – GFP</span>: This composite is designed so that transformed plants will have constitutive expression of green fluorescent protein. Having this constant expression provides the basis to measure any other inducible fluorescent proteins’ fluorescence relative to an internal control. </p><br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
<br />
<br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/CompositeTeam:Nevada/Composite2010-10-27T03:45:47Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Composite UNR.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
== Composite Parts ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*RD29A Promoter + Strong Plant Kozak (RBS) + RFP [[Team:Nevada/registry submissions]]<br />
*35S Promoter + (Strong Plant RBS + GFP) From K414001 [[Team:Nevada/registry submissions]]<br />
----<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;">RD29A – RFP</span>: This composite is designed so that transformed plants will yield red fluorescent protein under cold, drought, or high soil salinity conditions. We were successful in developing cold-responsive red fluorescent NT cells in time for the jamboree. Please check out the <html><a href="https://2010.igem.org/Team:Nevada/Results"><span style="color:#1569C7;font-weight:bold;">Transformation Results</span></a></html>.</p><br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;">35S – GFP</span>: This composite is designed so that transformed plants will constitutively express green fluorescent protein. Having this constant expression provides the basis to measure any other inducible fluorescent proteins’ fluorescents relative to an internal control. </p><br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
<br />
<br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/ResultsTeam:Nevada/Results2010-10-27T03:27:09Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Results UNR Final.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<br />
== NT Cell Transformation Results ==<br />
<br />
<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/Results" tabindex="2">Results</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/BY-2 (NT1)Transformation Protocol" tabindex="1">NT Cell Transformation Protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/Agrobacterium Transformations" tabindex="3">Agrobacterium Transformations</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/Transgenic Plants" tabindex="3">Transgenic Plants: into the Wild</a></li><br />
</ul><br />
</div><br />
</html><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG"><img src="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG" class="shadow" style="float:center;width:400px;margin:10px"></a><br />
</html><br />
'''Plated NT Cells Immediately after Transformation'''<br />
<br />
----<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/60/Igem_ntcells(2).jpg"><img src="https://static.igem.org/mediawiki/2010/6/60/Igem_ntcells(2).jpg" class="shadow" style="float:center;width:400px;margin:10px"></a><br />
</html><br />
'''Plated Transformed NT Cells after about 3 weeks of Cell Growth'''<br />
<br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/4/46/RD29A_Results.png"><img src="https://static.igem.org/mediawiki/2010/4/46/RD29A_Results.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
</html><br />
<p>'''RD29A + RFP Transformed NT Cells after about 3 weeks of growth:''' The NT cells shown above were transformed with RD29A+RFP, a composite part in which the RD29A promoter is turned on due to external stimuli such as cold, drought or salinity. When the RD29A promoter is turned "on" it then turns on its reporter, in this case RFP. After about 3 weeks of NT cell growth, the transformed cells were "cold stressed" for about 3hrs in 4 degrees Celsius. The results shown indicate that when cold stressed, the transformed NT cells become fluorescent with RFP. Over the next few weeks we hope to see this signal grow stronger and test these NT cells' responsiveness to other stresses that induce the RD29A promoter.</p><br />
<br><br />
----<br />
<br><br />
<p>'''35S + GFP Transformed NT Cells:''' Currently, our transformed NT cells that contain the composite part, 35S + GFP, have not grow enough to determine whether or not the transformation was successful. The 35S promoter was designed as a constitutive control and would always be turned "on", thus maintaining a constant GFP fluorescent signal. Over the next few weeks we hope to see more cell growth and eventually transformed cells that produce green fluorescent protein constitutively.</p><br />
<br><br />
<br />
----<br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/ResultsTeam:Nevada/Results2010-10-27T03:24:07Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Results UNR Final.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<br />
== NT Cell Transformation Results ==<br />
<br />
<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/Results" tabindex="2">Results</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/BY-2 (NT1)Transformation Protocol" tabindex="1">NT Cell Transformation Protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/Agrobacterium Transformations" tabindex="3">Agrobacterium Transformations</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/Transgenic Plants" tabindex="3">Transgenic Plants: into the Wild</a></li><br />
</ul><br />
</div><br />
</html><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG"><img src="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG" class="shadow" style="float:center;width:600px;margin:10px"></a><br />
</html><br />
<p>'''Plated NT Cells Immediately after Transformation'''</p><br />
<br />
----<br />
<html><a href="https://static.igem.org/mediawiki/2010/6/60/Igem_ntcells(2).jpg"><img src="https://static.igem.org/mediawiki/2010/6/60/Igem_ntcells(2).jpg" class="shadow" style="float:center;width:600px;margin:10px"></a><br />
</html><br />
<p>'''Plated Transformed NT Cells after about 3 weeks of Cell Growth'''</p><br />
<br />
----<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/4/46/RD29A_Results.png"><img src="https://static.igem.org/mediawiki/2010/4/46/RD29A_Results.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
</html><br />
<p>'''RD29A + RFP Transformed NT Cells after about 3 weeks of growth:''' The NT cells shown above were transformed with RD29A+RFP, a composite part in which the RD29A promoter is turned on due to external stimuli such as cold, drought or salinity. When the RD29A promoter is turned "on" it then turns on its reporter, in this case RFP. After about 3 weeks of NT cell growth, the transformed cells were "cold stressed" for about 3hrs in 4 degrees Celsius. The results shown indicate that when cold stressed, the transformed NT cells become fluorescent with RFP. Over the next few weeks we hope to see this signal grow stronger and test these NT cells' responsiveness to other stresses that induce the RD29A promoter.</p><br />
<br><br />
----<br />
<br><br />
<p>'''35S + GFP Transformed NT Cells:''' Currently, our transformed NT cells that contain the composite part, 35S + GFP, have not grow enough to determine whether or not the transformation was successful. The 35S promoter was designed as a constitutive control and would always be turned "on", thus maintaining a constant GFP fluorescent signal. Over the next few weeks we hope to see more cell growth and eventually transformed cells that produce green fluorescent protein constitutively.</p><br />
<br><br />
<br />
----<br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/TeamTeam:Nevada/Team2010-10-27T03:21:08Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Picture Team.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
<p style="text-align:left;"><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&spades;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&hearts;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&clubs;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&diams;</span>&nbsp;<span style="font-family:Arial;font-size:16pt;text-decoration:bold;color:#15317E;background-color:ffffff;">Meet the Team</span>&nbsp;<span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&diams;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&clubs;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&hearts;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&spades;</span><br />
</p><br />
----<br />
<div style="padding: 10px 0 10px 10px;"><br />
<br />
<html><br />
<a href="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG" class="shadow" style="float:left;margin:0px;width:600px"></a><br />
</html><br />
<br />
<p style="text-align:center;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Advisors</span><br />
</p><br />
----<br />
<br />
[[Image:Christie.jpg|center|thumb|alt=alt text|<p style="text-align:center;"><span style="font-family:Times;font-size:12pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Dr. Christie Howard</span></p>]]<br />
<br />
[[Image:Shintani.jpg|center|thumb|alt=alt text|<p style="text-align:center;"><span style="font-family:Times;font-size:12pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Dr. David Shintani</span></p>]]<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Undergraduates</span><br />
</p><br />
----<br />
<gallery widths=170px heights=170px perrow=4><br />
Image:Hilary UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span></p><br />
Image:mpolasko2.JPG|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span></p><br />
Image:Randy.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span></p><br />
Image:Copley.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span></p><br />
Image:Bryson UNR 2.png| <p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span></p><br />
Image:Gladwill.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span></p><br />
Image:Image-Elaine.jpeg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span></p><br />
Image:Sam final UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span></p><br />
&nbsp;<br />
Image:Nick UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span></p><br />
Image:Hileary.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span></p><br />
</gallery><br />
<br />
<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:normal;text-decoration:bold;color:#15317E;background-color:ffffff;">What We Did</span><br />
</p><br />
----<br />
<br />
<div style="padding:10px;clear:both"><br />
{| border="0" cellpadding="0" cellspacing="20"<br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Team President</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">NT Cell Transformations</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter + Plant Kozak + RFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + EYFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + mCherry</span><br />
*<span style="font-size:12pt;font-weight:bold;">Composite - 35s Promoter + Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;"> DREB1C Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">35S Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Modeling</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">RD29A Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|&nbsp;<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span><br />
|-<br />
|&nbsp;<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|}<br />
----<br />
<br />
</div><br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:NevadaTeam:Nevada2010-10-27T03:06:09Z<p>Mpolasko: </p>
<hr />
<div>{{nevadamain}}<br />
== Abstract ==<br />
<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/8/8b/IGEM_Team_2010.jpg/400px-IGEM_Team_2010.jpg" class="shadow" style="float:right"><br />
</html><br />
<br />
<br><br />
<p>Our long term goals are to develop plant biosensors that reliably measure changes in the local environment. In the process of developing biosensors, the''' 2010 Nevada iGEM Team''' need to develop fundamental promoters, reporters, and plasmids for plants. Therefore, 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 (NT cells) as a model for the expression of higher plant genes for future iGEM competitions. NT cells are a faster, cheaper, and safer model than traditional plant transformation. 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><br />
<br><br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png"><img src="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
</html><br />
<p style="text-align:right;"><span style="font-size:10px;">Fluorescent Plant Image Taken From: http://www.edinformatics.com/inventions_inventors/genetic_engineering.htm</span></p><br />
<br />
----<br />
<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/plant_compatible_reportersTeam:Nevada/plant compatible reporters2010-10-26T05:20:34Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:KozakReporter UNR.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
== Reporters ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*Strong Plant RBS (Kozak sequence) + GFP from E0040 [[Team:Nevada/registry submissions]]<br />
*Strong Plant RBS (Kozak sequence) + EYFP from E0030 [[Team:Nevada/registry submissions]]<br />
*Strong Plant RBS (Kozak sequence) + mCherry from J06504 [[Team:Nevada/registry submissions]]<br />
----<br />
<br />
<html><br />
<a href="https://static.igem.org/mediawiki/2010/7/7e/Reporters.png"><img src="https://static.igem.org/mediawiki/2010/7/7e/Reporters.png" class="shadow" style="float:left;width:450px;margin:10px"></a><br />
</html><br />
<br />
<br />
<p style="text-align:center;"><span style="text-decoration:underline;font-weight:bold">Building Consensus</span></p><br />
<br />
While all the aforementioned issues are important, the aspect of plant engineering that we believe is fundamental for future iGEM teams to consider is the ribosome binding site (RBS). RBS can differ between species, but it varies widely between eukaryotes, such as yeast, animals, and plants. The term RBS can be misleading because ribosomes can weakly associate with RNA as it “scans” along the sequence. Why is the ribosome “scanning?” Ribosomes initiate translation, and the “start” site that we have all been taught for eukaryotes is the methionine sequence AUG (or ATG if you are biased towards DNA). However, almost thirty years ago, a researcher named Kozak discovered that it is not simply AUG which initiates translation but the context of that AUG, the surrounding sequence, influenced whether translation actually began with one AUG sequence versus another. These context sequences, as they have been discovered in different organisms, eventually have been named Kozak sequences. <br />
<br />
Even among plants, there can be different Kozak sequences. Where we decided to contribute to iGEM was to supply the registry with the first fluorescent proteins with plant compatible RBS or Kozak sequences. We have chosen a generically ‘strong’ Kozak sequence that should provide the maximum translational efficiency for dicots, but it should also work generally well enough in most if not all plants. Our sequence is AAA AAA AAA ACA upstream of the AUG. An important aspect of Kozak sequences one should consider is there are both an upstream component and a downstream component. The string of purines upstream is associated with many plant Kozak sequences, but almost equally important is to have a G at the +4 position, or immediately following the AUG. Therefore, AAAAAAAAACA'''AUG'''G is likely to have the highest translational efficiency. Fortunately, two of the florescent proteins, EYFP and mCherry have this context. GFP, however does not. It is missing the G at +4 which will hurt its translational efficiency. Instead, a C occupies that position which codes for arginine, R. There is no codon for arginine that starts with G. Unless a known mutation can be made, we may stuck with that hindrance. However, we have attempted to compensate in one of our composite parts, 35S GFP. 35S is a constitutive plant promoter. Ideally, the high transcriptional activity can compensate for the weakened translational efficiency. <br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
----<br />
<p style="text-align:center;"><span style="text-decoration:underline;font-weight:bold">Engineering Possibilities (Fine tuning your translation)</span></p><br />
As we see it, there are three ways future iGEM teams could engage the plant Kozak sequence to modify gene expression in plants: identity, distance, or deking.<br />
<br />
'''1)Identity''': The most obvious way of affecting translational efficiency would be to alter the Kozak sequences. Having genes each prefaced with the same promoter but with different Kozak contextual sequences would tier the levels of expression. One could have an optimum Kozak like the one we have submitted and also engineer a weaker Kozak sequence for another gene which has relatively 50% expression compared to the optimum gene expressed. Consulting literature or experimenting in less-frequently researched plants will allow for greater variability in controlling expression. <br />
<br />
'''2)Distance''': Another way to affect your protein expression would be how far the ‘true’ Kozak sequence is relative to the 5’ cap. A strong Kozak sequence means nothing if it is several hundred base pairs away from the end of the promoter. Because our team wanted to supply genes with maximal expression, our parts are intended to be placed immediately behind the promoter. Yet, engineering plasmids that put gaps between the promoter and actual Kozak, or primers designed to put more space in between the promoter and start site, could also be one way of dialing the levels of expression. <br />
<br />
'''3)Deking''':(Realizing a team from a desert is using a hockey term): The “fake out.” A third alternative that combines the principles of identity and distance is to create one, two, or a few pseudo-start sites. Psuedo-start sites means one would engineer AUG sequences upstream of the actual, desired one. These sequences would be in a poorer context and/or would translate into little nonsense peptides that theoretically have no function. Think of them as siAUG (short interfering AUG sites). These fake sites would knockdown expression. <br />
<br />
In Summary, Kozak sequences have plenty of promise in the engineering side of iGEM, but Kozak sequences are also a necessity that all iGEM teams must consider if they are to express proteins in plants.<br />
<br />
<span style="text-decoration:underline;font-weight:bold">References</span><br />
<br />
'''Agarwal, S., Jha, S., Sanyal, I., Amla, D.V.''' (2009) Effect of point mutations in translation initiation context on the expression of recombinant human alpha1-proteinase inhibitor in transgenic tomato plants. ''Plant Cell Reports''. 28: 1791-1798.<br />
<br />
'''Joshi, C.P., Zhou, H., Huang, X., Chiang, V.L.''' (1997) Context sequences of translation initiation codon in plants. ''Plant Molecular Biology''. 35: 993-1001.<br />
<br />
'''Kozak, M.''' (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribososomes. ''Cell''. 44: 283-92.<br />
<br />
'''Matsuda, D., Dreher, T.W.''' (2006) Close spacing of AUG initiation codons confers dicistronic character on a eukaryotic mRNA. ''RNA''. 12: 1138-1349.<br />
<br />
<br />
<br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/Transgenic_PlantsTeam:Nevada/Transgenic Plants2010-10-26T05:19:42Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Transgenic Plants.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<br />
== Transgenic Plants: into the Wild ==<br />
<br />
<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/Results" tabindex="2">Results</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/BY-2 (NT1)Transformation Protocol" tabindex="1">NT Cell Transformation Protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/Agrobacterium Transformations" tabindex="3">Agrobacterium Transformations</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/Transgenic Plants" tabindex="3">Transgenic Plants: into the Wild</a></li><br />
</ul><br />
</div><br />
</html><br />
<br />
<br />
<p style="text-align:center;"><span style="text-decoration:underline;font-weight:bold">Do You Have A Green Thumb?</span></p><br />
<p>The Nevada iGEM team along with a handful of other teams this year are looking to expand iGEM to the world of plants. iGEM will finally have a medium to explore eukaryotic gene manipulation without requiring yeast farms or human experimentation. Plants provide a kingdom of exploration that could make iGEM a potential attractant to commercial biotech firms as it brings the possibilities of synthetic biology out into the open from the patents and trade secret shadows which a lot of firms rely on today.</p> <br />
<br />
<p>Engaging a whole new realm of organisms requires fundamental understanding of those organisms and how they differ from the current models. That understanding from iGEM teams is essential to its success. Obviously, plants come with their own unique promoters, activators, and repressors with which to play. We added a few plant promoters to the iGEM registry for future teams to use. Also, there will need to be new consideration as to what types of proteins with which we can transform the plants. Fortunately, some essential eukaryotic proteins that iGEM has dealt with, like the fluorescent proteins, have already been used in plant experimentation.</p><br />
<br />
As far as testing new models, we believe our NT cells may provide a cheaper, faster alternative to teams interested in experimenting with plants. Also, the NT cells are safer to use and pose less risk to the environment then experimenting with actual plants. <br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/plant_compatible_reportersTeam:Nevada/plant compatible reporters2010-10-26T05:19:07Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:KozakReporter UNR.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
== Reporters ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*Strong Plant RBS (Kozak sequence) + GFP from E0040 [[Team:Nevada/registry submissions]]<br />
*Strong Plant RBS (Kozak sequence) + EYFP from E0030 [[Team:Nevada/registry submissions]]<br />
*Strong Plant RBS (Kozak sequence) + mCherry from J06504 [[Team:Nevada/registry submissions]]<br />
----<br />
<br />
<html><br />
<a href="https://static.igem.org/mediawiki/2010/7/7e/Reporters.png"><img src="https://static.igem.org/mediawiki/2010/7/7e/Reporters.png" class="shadow" style="float:left;width:450px;margin:10px"></a><br />
</html><br />
<br />
<br />
<p style="text-align:center;"><span style="text-decoration:underline;font-weight:bold">Building Consensus</span></p><br />
<br />
While all the aforementioned issues are important, the aspect of plant engineering that we believe is fundamental for future iGEM teams to consider is the ribosome binding site (RBS). RBS can differ between species, but it varies widely between eukaryotes, such as yeast, animals, and plants. The term RBS can be misleading because ribosomes can weakly associate with RNA as it “scans” along the sequence. Why is the ribosome “scanning?” Ribosomes initiate translation, and the “start” site that we have all been taught for eukaryotes is the methionine sequence AUG (or ATG if you are biased towards DNA). However, almost thirty years ago, a researcher named Kozak discovered that it is not simply AUG which initiates translation but the context of that AUG, the surrounding sequence, influenced whether translation actually began with one AUG sequence versus another. These context sequences, as they have been discovered in different organisms, eventually have been named Kozak sequences. <br />
<br />
Even among plants, there can be different Kozak sequences. Where we decided to contribute to iGEM was to supply the registry with the first fluorescent proteins with plant compatible RBS or Kozak sequences. We have chosen a generically ‘strong’ Kozak sequence that should provide the maximum translational efficiency for dicots, but it should also work generally well enough in most if not all plants. Our sequence is AAA AAA AAA ACA upstream of the AUG. An important aspect of Kozak sequences one should consider is there are both an upstream component and a downstream component. The string of purines upstream is associated with many plant Kozak sequences, but almost equally important is to have a G at the +4 position, or immediately following the AUG. Therefore, AAAAAAAAACA'''AUG'''G is likely to have the highest translational efficiency. Fortunately, two of the florescent proteins, EYFP and mCherry have this context. GFP, however does not. It is missing the G at +4 which will hurt its translational efficiency. Instead, a C occupies that position which codes for arginine, R. There is no codon for arginine that starts with G. Unless a known mutation can be made, we may stuck with that hindrance. However, we have attempted to compensate in one of our composite parts, 35S GFP. 35S is a constitutive plant promoter. Ideally, the high transcriptional activity can compensate for the weakened translational efficiency. <br />
----<br />
<p style="text-align:center;"><span style="text-decoration:underline;font-weight:bold">Engineering Possibilities (Fine tuning your translation)</span></p><br />
As we see it, there are three ways future iGEM teams could engage the plant Kozak sequence to modify gene expression in plants: identity, distance, or deking.<br />
<br />
'''1)Identity''': The most obvious way of affecting translational efficiency would be to alter the Kozak sequences. Having genes each prefaced with the same promoter but with different Kozak contextual sequences would tier the levels of expression. One could have an optimum Kozak like the one we have submitted and also engineer a weaker Kozak sequence for another gene which has relatively 50% expression compared to the optimum gene expressed. Consulting literature or experimenting in less-frequently researched plants will allow for greater variability in controlling expression. <br />
<br />
'''2)Distance''': Another way to affect your protein expression would be how far the ‘true’ Kozak sequence is relative to the 5’ cap. A strong Kozak sequence means nothing if it is several hundred base pairs away from the end of the promoter. Because our team wanted to supply genes with maximal expression, our parts are intended to be placed immediately behind the promoter. Yet, engineering plasmids that put gaps between the promoter and actual Kozak, or primers designed to put more space in between the promoter and start site, could also be one way of dialing the levels of expression. <br />
<br />
'''3)Deking''':(Realizing a team from a desert is using a hockey term): The “fake out.” A third alternative that combines the principles of identity and distance is to create one, two, or a few pseudo-start sites. Psuedo-start sites means one would engineer AUG sequences upstream of the actual, desired one. These sequences would be in a poorer context and/or would translate into little nonsense peptides that theoretically have no function. Think of them as siAUG (short interfering AUG sites). These fake sites would knockdown expression. <br />
<br />
In Summary, Kozak sequences have plenty of promise in the engineering side of iGEM, but Kozak sequences are also a necessity that all iGEM teams must consider if they are to express proteins in plants.<br />
<br />
<span style="text-decoration:underline;font-weight:bold">References</span><br />
<br />
'''Agarwal, S., Jha, S., Sanyal, I., Amla, D.V.''' (2009) Effect of point mutations in translation initiation context on the expression of recombinant human alpha1-proteinase inhibitor in transgenic tomato plants. ''Plant Cell Reports''. 28: 1791-1798.<br />
<br />
'''Joshi, C.P., Zhou, H., Huang, X., Chiang, V.L.''' (1997) Context sequences of translation initiation codon in plants. ''Plant Molecular Biology''. 35: 993-1001.<br />
<br />
'''Kozak, M.''' (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribososomes. ''Cell''. 44: 283-92.<br />
<br />
'''Matsuda, D., Dreher, T.W.''' (2006) Close spacing of AUG initiation codons confers dicistronic character on a eukaryotic mRNA. ''RNA''. 12: 1138-1349.<br />
<br />
<br />
<br />
<br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
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!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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<div>{{nevadaDREB1C}}<br />
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== Promoters ==<br />
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<h1>Subpages</h1><br />
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<li><a href="https://2010.igem.org/Team:Nevada/DREB1C" tabindex="1">DREB1C</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/RD29A" tabindex="2">rd29A</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/35S" tabindex="3">35S</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/CD2Inducible" tabindex="4">CD2+ Inducible</a></li><br />
</ul><br />
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</html><br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png '''DREB1C promoter''' [[Team:Nevada/registry submissions]] <br />
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<p><html><a href="https://static.igem.org/mediawiki/igem.org/d/d1/Chris_Igem.jpg"><img src="https://static.igem.org/mediawiki/igem.org/d/d1/Chris_Igem.jpg" class="shadow" style="float:left;width:200px;margin:10px"></a><br />
</html> The <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html> iGEM promoter is derived from a transcription factor that is up-regulated by cold stress and down-regulated by circadian controls to prevent plant growth retardation due to the buildup of <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html> (Dehydration Response Element Binding Protein) during the day (the cause of dwarfism). The promoter region for <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html> begins just upstream of the translational start sequence (ATG) and contains 463 bp of the upstream sequence with six ADA independent, <i>cis</i>-acting elements for the up-regulation during cold stress and circadian controlled down-regulation.</p><br />
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<p>The <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html> iGEM promoter was isolated using custom primers directly from the <i>Arabidopsis thaliana</i> genome via PCR, blunt end Topo cloned, and then ligated to pSB1C3 using EcoR1 and Pst1 sites.</p><br />
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<br>'''References'''<br><br />
'''Kazuo Nakashima and Kazuko Yamaguchi-Shinozakia''', Regulons involved in osmotic stress-responsive and cold stress-responsive gene expression in plants, Physiologia Plantarum 126: 62–71. 2006<br />
'''Satoshi Kidokoro, Kyonoshin Maruyama, Kazuo Nakashima, Yoshiyuki Imura2, Yoshihiro Narusaka3, Zabta K. Shinwari4, Yuriko Osakabe, Yasunari Fujita, Junya Mizoi, Kazuo Shinozaki, and Kazuko Yamaguchi-Shinozaki''', The Phytochrome-Interacting Factor PIF7 Negatively Regulates DREB1 Expression under Circadian Control in Arabidopsis, Plant Physiol. Vol. 151, 2009<br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
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!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/fundraising_sponsorshipsTeam:Nevada/fundraising sponsorships2010-10-26T05:15:10Z<p>Mpolasko: </p>
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<div>{{Nevada_css}}<br />
[[Image:UNR Funraising.png|border|left|900px]]<br />
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== Fundraising and Sponsorships ==<br />
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<html><a href="https://static.igem.org/mediawiki/2010/9/9b/12859_544382805569_23800361_31674393_4924904_n.jpg"><img src="https://static.igem.org/mediawiki/2010/9/9b/12859_544382805569_23800361_31674393_4924904_n.jpg" class="shadow" style="float:left;width:300px;margin:10px"></a></html><p>In order for the Nevada iGEM team to complete the necessary Bio Bricks for competition, a large source of financial capital was needed. It is a result of the generosity of Nevada INBRE that a majority of the tools and equipment (including, but not limited to, oligonucleotides, plasmids, cell lines, enzymes, and buffers) were met in a timely manner. However, funding was also needed to pay for the necessary expenses of travel, lodgings, and meals for a team of 10 individuals to the Jamboree in Boston, MA. It was out of a desperation that the iGEM team turned to the Reno community for help and support. Through cooperative efforts and local generosity, the Nevada team hosted a series of bar crawls, drink specials, ice cream socials, and concerts in and around the downtown Reno community. Through the selection of strategic locations and dates during summer and fall, these fundraising efforts helped attract business to the local economy and spark the interest of young scientists in and around the University community. The feedback and support was overwhelmingly positive. The events were successful in not only raising money, but also raising awareness about synthetic biology and the University of Nevada’s growing biotechnology research. Thanks to the community’s efforts, the Nevada team was able to raise over $6,000! In a state devastated by high unemployment and low revenue from tourism, the support and generosity that was received was incredible. It is thanks to the efforts of local entrepreneurs in the community’s entertainment district and university students that allowed the team to raise the necessary money to help alleviate the costs of travel.</p><br />
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'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $6,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Mpolaskohttp://2010.igem.org/Team:NevadaTeam:Nevada2010-10-26T05:14:40Z<p>Mpolasko: </p>
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<div>{{nevadamain}}<br />
<p>&nbsp;</p><br />
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== Abstract ==<br />
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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 (NT cells) 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. 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><br />
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<html><a href="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png"><img src="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
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'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $6,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/registry_submissionsTeam:Nevada/registry submissions2010-10-26T05:13:48Z<p>Mpolasko: </p>
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<div>{{Nevada_css}}<br />
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<p>&nbsp;</p><br />
== Registry Submissions ==<br />
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<groupparts>iGEM010 Nevada</groupparts><br />
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<br />
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<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $6,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/RD29APromoterTeam:Nevada/RD29APromoter2010-10-26T05:06:32Z<p>Mpolasko: </p>
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<div>{{Nevada_css}}<br />
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==rd29A Stress Induced Promoter==<br />
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<html><br />
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<p>The stress-inducible rd29A (responsive to dehydration 29A) promoter is derived from Arabidopsis thaliana, which is a small flowering plant that is a member of the mustard family (Brassicaceae). DRE and ABA dependent binding sequences have been found within the promoter region, and are shown to be independent of each other. The DRE/CRT binding sequences are the target of the binding protein <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html>. <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html> activation of rd29A is relegated by cold stress responses, yet salinity and drought stresses also activate rd29A using alternative ABA independent and dependent transcription factors.</p><br />
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<p>From previous studies, the rd29A promoter not only increases the resistance to different stresses in plants, but it also minimizes the negative effects such as dwarfism (only 30% grown reduction compared to the wild type plant and only a slight growth retardation phenotype on the tobacco plant ) in comparison to the 78% growth reduction of the 35S promoter. This proved that the rd29A is a better promoter than the 35S when used with a stress reporter. Another study also showed that the survival rates of the transgenic clones (with the rd29A promoter) had a greater probability of survival after recovery from exposure to freezing temperatures. This was compared to the non-transgenic cells that showed damage to the plant with no recovery after freezing. Therefore, the rd29A promoter is critically important for it can potentially improve agricultural techniques that farmers can use for their crops. </p><br />
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<br><p>'''References'''<br><br />
'''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.<br />
<br><br />
'''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.</div>Mpolaskohttp://2010.igem.org/Team:Nevada/DREB1CPromoterTeam:Nevada/DREB1CPromoter2010-10-26T05:05:54Z<p>Mpolasko: </p>
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==DREB1C: Cold Induced Stress Promoter==<br />
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<p>Plants experience a wide range of abiotic stresses due to their sessile nature. Plants have evolved many biochemical responses to stresses that range from cold and drought to salinity and osmotic factors. Substantial research has revealed a complex network of genes that work together during stress conditions. A key player in the abiotic stress response in Arabidopsis is DREB1 (Dehydration Response Element Binding Protein) a family of three related (A, B, C) transcription factors that bind to DRE (Dehydration Response Element), a <i>cis</i>-acting element that functions in ABA-independent gene expression, specifically as an up regulator of rd29A. Researchers have demonstrated that <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter"><span style="color:#1569C7;font-weight:bold;">rd29A</span></a></html> is up regulated in response to salt, drought and cold conditions (Shinozaki, 1998).</p><br />
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<p>Plant cold stress responses in Arabidopsis have been shown to contain several pathways that are specific to abiotic stresses. The DREB1 family has been shown to be most strongly expressed in response to cold stress, salinity, osmotic, and drought stresses. Specifically, DREB1C has been shown to be most active in cold stress response, while DREB1A and DREB1B show expression patterns in drought and salinity as well (Yamaguchi-Shinozaki, 2009). Promoter regions for the three DREB1 proteins contain six homologous sequences, or boxes, that correspond to specific binding motifs. While the specific mechanism of cold induction is not yet clear, DREB1C promoter analysis has shown MYB, and MYC binding sequences(Shinozaki, 1998)as well as evidence that the CAMTA3, calcium dependent protein, is involved in up regulation of the cold response (Thomashow, 2009).</p><br />
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<p>While DREB1C up regulation shows significant increase in plant cold tolerance, an over expression results in dwarfism and stunted growth. It has been shown that PIF7, a Phytochrome interacting factor (PIF) which is integral to circadian rhythm controls, act as a daytime inhibitor of DREB1C (Yamaguchi-Shinozaki, 2009). This helps mitigate damage from over expression by acting as one of many counter regulatory elements.</p><br />
<p>These genes have also been shown to have orthologs in rice and maize, OsDREB1A and ZmDREB1A respectively. Expression of these gene constructs in transgenic Arabidopsis showed increased tolerance to cold, salt, and drought stresses. While these genes do not bind all target DREB1 sequences, they show expression patterns that mimic Arabidopsis (Yamaguchi-Shinozaki, 2006).</p><br />
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<p>The DREB1C promoter could be combined with a reporter gene to serve as a valuable biosensor for cold induced stresses. The fact that it is up regulated by cold stress (MYB and MYC binding motifs) (Shinozaki, 1998) but down regulated by circadian controls (PIF7 binding) (Yamaguchi-Shinozaki, 2009) allow for a more accurate warning when particular plants were under cold stress while keeping constitutive levels low. Evidence that it shares expression orthologs in some plants hints that the DREB1C promoter could be adapted to fit many crop types.</p><br />
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</div><br />
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<br><p>'''References'''<br><br />
<b>Zabta K. Shinwari, Kazuo Nakashima, Setsuko Miura, Mie Kasuga, Motoaki Seki, Kazuko Yamaguchi-Shinozaki, and Kazuo Shinozaki</b>, An Arabidopsis Gene Family Encoding DRE/CRT Binding Proteins Involved in Low-Temperature-Responsive Gene Expression, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 250, 161–170 (1998)</p><br />
<p><b>Colleen J. Doherty, Heather A. Van Buskirk, Susan J. Myers, and Michael F. Thomashowa</b>, Roles for Arabidopsis CAMTA Transcription Factors in Cold-Regulated Gene Expression and Freezing Tolerance, The Plant Cell, Vol. 21: 972–984, March 2009</p><br />
<p><b>Kazuo Nakashima and Kazuko Yamaguchi-Shinozaki</b>, Regulons involved in osmotic stress-responsive and cold stress-responsive gene expression in plants, Physiologia Plantarum 126: 62–71. 2006</p><br />
<p><b>Satoshi Kidokoro, Kyonoshin Maruyama, Kazuo Nakashima, Yoshiyuki Imura2, Yoshihiro Narusaka, Zabta K. Shinwari, Yuriko Osakabe, Yasunari Fujita, Junya Mizoi, Kazuo Shinozaki, and Kazuko Yamaguchi-Shinozaki</b>, The Phytochrome-Interacting Factor PIF7 Negatively Regulates DREB1 Expression under Circadian Control in Arabidopsis, Plant Physiol. Vol. 151, 2009</p></div>Mpolaskohttp://2010.igem.org/Team:Nevada/promotersTeam:Nevada/promoters2010-10-26T05:03:57Z<p>Mpolasko: /* Promoters */</p>
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== Promoters ==<br />
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<html><br />
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<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/DREB1C" tabindex="1">DREB1C</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/RD29A" tabindex="2">rd29A</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/35S" tabindex="3">35S</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/CD2Inducible" tabindex="4">CD2+ Inducible</a></li><br />
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<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><br />
</html><p>The Nevada team has created environmental stress sensors by using <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter"><span style="color:#1569C7;font-weight:bold;">rd29A</span></a></html> and <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html> promoters to express red fluorescent protein and other bio-fluorescent markers. When induced by environmental stress, plants carrying these genes can easily be detected by the farmer walking through his field or by a plane flying over acres of farmland. Promoter elements are short and easy to work with. They also allow for modification and specialization. <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html> and <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter"><span style="color:#1569C7;font-weight:bold;">rd29A</span></a></html> both have multiple binding motifs in their promoter regions allowing for variation in expression levels and the particular stresses that induce them. 35s is a constitutive promoter that can be valuable for control groups in stress and other plant response research.</p><br />
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!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/promotersTeam:Nevada/promoters2010-10-26T05:00:55Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Promoters UNR Final.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<br />
<p>&nbsp;</p><br />
<br />
== Promoters ==<br />
<br />
<br />
<br />
<br />
<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/DREB1C" tabindex="1">DREB1C</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/RD29A" tabindex="2">RD29A</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/35S" tabindex="3">35S</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/CD2Inducible" tabindex="4">CD2+ Inducible</a></li><br />
</ul><br />
</div><br />
</html><br />
<br />
<html><br />
<body link=#00ffff><br />
<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><br />
</html><p>The Nevada team has created environmental stress sensors by using <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter"><span style="color:#1569C7;">RD29A</span></a></html> and <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;">DREB1C</span></a></html> promoters to express red fluorescent protein and other bio-fluorescent markers. When induced by environmental stress, plants carrying these genes can easily be detected by the farmer walking through his field or by a plane flying over acres of farmland. Promoter elements are short and easy to work with. They also allow for modification and specialization. <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;">DREB1C</span></a></html> and <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter"><span style="color:#1569C7;">RD29A</span></a></html> both have multiple binding motifs in their promoter regions allowing for variation in expression levels and the particular stresses that induce them. 35s is a constitutive promoter that can be valuable for control groups in stress and other plant response research.</p><br />
<br />
<br />
<p>&nbsp;</p><br />
<br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/TransformationsTeam:Nevada/Transformations2010-10-25T23:12:24Z<p>Mpolasko: /* NT Cell Transformations */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:NT cell transformations.png|border|left|950px]]<br />
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{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<br />
== NT Cell Transformations ==<br />
<br />
<br />
<p>This year the Nevada iGEM team has decided to utilize Tobacco (''Nicotiana tabacum'') NT1 cells due to its vigorous ability to grow rapidly as well as its simple and convenient model system characteristics. The original idea was to test the validity of utilizing Nicotiana tabacum (NT cells) as a model for the expression of higher plant genes for future iGEM competitions. Though the utilization of ''Arabidopsis'' was possible, we aimed to make a quick proof-of-concept test model that other iGEMers could utilize before moving synthetic constructs into plants of interest. This model system was specifically used to make the evaluation of our plant expression genes quick, simple and efficient. Typical whole-plant systems can take anywhere from a year or more to obtain results however the NT1 model system allowed us to obtain transformants on solid media in less than 5 weeks. The NT1 cell model system is also known to facilitate protein production and avoids typical complications of alternative ''in planta'' production – features highly advantageous to any type of research. Though this model system is new and unexplored in the iGEM areana, we found it to be extremely proficient and promising in terms of results.</p><br />
<br />
----<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG"><img src="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG" class="shadow" style="float:left;width:500px;margin:10px"></a><br />
</html><br />
<br />
<br />
<p><span style="font-size:18px;font-weight:bold;text-decoration:underline">NT Cell Protocol</span></p><br />
<br />
<p>'''CARE OF BY-2 (NT1) CELL CULTURES'''</p><br />
(adapted from a letter by Dr. Michael Sullivan) <br />
To readapt a culture on plates, simply transfer some of the cells back into liquid <br />
media. We usually pipette the cell suspension up and down to break up any clumps. It <br />
may be best to start out with a smaller culture volume when you first go back to liquid; <br />
BY-2 seems to be a bit happier if it isn't seeded to thinly. Allow the culture to grow <br />
until it is the consistency of thin applesauce. At this point, you should be able to go to <br />
a 50 ml culture and start subculturing as described below. In our experience, wild-type <br />
BY-2 readapts quickly to liquid to give a smooth culture; transformed lines tend to be more <br />
variable, with some producing smooth cultures, some producing clumpy ones, and some going <br />
back and forth between these two states. We've found that clumpy cultures do not interfere <br />
with our half-live measurements, although manipulating them can be a bit more difficult. <br />
<br />
We grow our liquid cultures in 50 ml of media in 250 ml baffle flasks at 28 degrees C <br />
with gentle shaking (150 rpm). Using a baffle flask does not seem to be a requirement, many <br />
people grow these cells in regular flasks with no problem. We subculture them once a week <br />
by transferring 5% of the culture to fresh media. We generally maintain two flasks <br />
(in two separate shakers) of two separate subcultures (one subcultured Monday, one on Friday) <br />
in case one of the cultures crashes. Also, you can maintain the culture on a plate. <br />
Note that, for liquid cultures of transformed lines, we usually use a smaller culture volume, <br />
e.g. 10 ml, for convenience. <br />
<br />
<br />
'''NT KC MEDIA - LIQUID OR PLATES<br />
NT LIQUID:''' <br />
(amounts are for 1L of media): <br />
750 ml H2O <br />
4.3 g MS salts (add slowly to liquid) <br />
30 g Sucrose <br />
50 ml 20X MES pH 5.7 <br />
10 ml B1 -inositol <br />
3 ml Miller's I <br />
10 ml 2,4-D, 10-4 M <br />
pH to 5.7 with 0.1 N KOH <br />
Bring volume to 1000ml <br />
Autoclave <br />
<br />
'''SOLID MEDIA:''' <br />
For plates only: Add to flasks 7 g/ L Phytagar before autoclaving <br />
Add kanamycin (100 mg/ml) <br />
Add carbenicillin (250 mg/ml) <br />
<br />
'''MEDIA COMPONENTS:''' <br />
Miller's I: 60 g KH2 PO4 per liter <br />
20 X MES: 10 g MES per liter, pH to 5.7 with 1N KOH <br />
B1 - Inositol: 0.1 g Thiamine, 10.0 g myo-inositol, H2O to final volume of 1 liter <br />
<br />
<br />
<br />
<p><span style="font-size:18px;font-weight:bold;text-decoration:underline">Transformation Protocol</span></p><br />
<p>'''BY-2 (NT1) Cell Transformation with Agrobactrium'''</p><br />
<p>'''Day 1:''' </p><br />
<p>1. Grow up 1 ml of the Agrobacterium overnight in LB + all selective drugs. This culture may be started from frozen glycerol cultures if necessary. </p><br />
<br />
<p>'''Day 2:''' </p><br />
<p>2. NT cells are used 3 days after splitting the NT cell culture. 4 ml of NT cells are required for each transformation with an additional 4 ml for the control culture which receives no bacteria. </p><br />
<br />
3. 1 ul Acetosyringone (20 mM in ethanol) is added per ml of NT cells. Typically treat the whole 50 ml culture at this point and discard any that is left over when I'm finished. <br />
<br />
4. Using a 10 ml pipette, the NT cells are pipetted in and out about 20 times. This helps to induce small lesions in the cells and increases the efficiency of the transformation. <br />
<br />
5. 75 ul of bacteria (dense growth) or 100 ul (moderate growth) are added to a petri dish containing 4 ml of NT cells (from step 4) and mixed thoroughly. REMEMBER TO INCLUDE A CONTROL HAVING NO BACTERIA. <br />
<br />
6. Wrap plates with parafilm and incubate for 3 days at 28 degrees C. <br />
<br />
<p>'''Day 5:''' </p><br />
7. To each plate add 10 ml of NT liquid medium containing 500 (u)g/ml carbenicillin (NTC).<br />
<br />
8. Collect cells off of the plates into 50 ml conical tubes and fill with NTC. <br />
<br />
9. Centrifuge at 1K for 4 minutes at room temperature in a swinging bucket rotor. <br />
<br />
10. Aspirate off liquid using pipet capped with a sterile blue 1 ml top. Change tip for each sample.<br />
<br />
11. Resuspend in 50 ml NTC and repeat spin. <br />
<br />
12. Repeat step 10 and 11 - 1 or 2 times. Strains that are fairly sensitive to carb require fewer washes. <br />
<br />
13. Resuspend cells in approximately 5 ml NTC and plate 2.0 ml on 2 NTKC (kanamycin 100 ug/ml carbenicillin 500 ul/ml) plates. When there is no longer lots of fluid on the plates (leave the plates open in the hood a few minutes of necessary), wrap them in parafilm.<br />
<br />
14. Incubate at 28 degrees C. Transformants should be large enough to transfer to fresh NTKC plates in 3-4 weeks. <br />
<br />
'''Supplies for each transformation (Remember the controls):''' <br />
<br />
Day 1 Supplies: <br />
LB + appropriate drugs<br />
Agrobacterium containing plasmid for transformation <br />
<br />
Day 2 Supplies: <br />
1 ml Agrobacterium overnight culture <br />
4 ml BY-2 cells - 3 days post subculture <br />
4 ul acetosyringone (20 mM) found in the (-20) refrigerator freezer <br />
pipets and pipetman <br />
1 petri plate <br />
<br />
Day 5 Supplies: <br />
200 ml NTC liquid <br />
50 ml conical tube <br />
swinging bucket centrifuge at room temp aspiration setup with 5 ml pipet capped with 1 ml blue tip <br />
2 NTKC plates <br />
pipetmen and tips <br />
<br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/4/4f/IMG_6028.JPG"><img src="https://static.igem.org/mediawiki/2010/4/4f/IMG_6028.JPG" class="shadow" style="float:left;width:430px;margin:10px"></a><br />
</html><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/3/3d/IMG_6035.JPG"><img src="https://static.igem.org/mediawiki/2010/3/3d/IMG_6035.JPG" class="shadow" style="float:right;width:430px;margin:10px"></a></html><br />
<br />
<br />
<br />
----<br />
<p>'''Left:''' NT Cell Culture after about 5 days. '''Right:''' NT Cell Transformation with RD29A (Cold, Drought, Salt) Inducible Promoter.</p><br />
<br />
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<!--- The Mission, Experiments ---><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-25T05:44:52Z<p>Mpolasko: </p>
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<div>{{Nevada_css}}<br />
[[Image:UNR Notebook.png|border|left|950px]]<br />
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{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
</p><br />
----<br />
==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF<br />
** Analysis of sequence of GFP (PSB1C3)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product - Bands showed at 500 bp<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
** Bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Topocloned PCR product of RD29A<br />
** Streaked single colonies of RD29A (Topo Vector)<br />
** Cell cultured single colonies of RD29A (Topo Vector)<br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
----<br />
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!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/SafetyTeam:Nevada/Safety2010-10-25T05:44:07Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Safety.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
== Safety ==<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/cd/IMG_6042.JPG"><img src="https://static.igem.org/mediawiki/2010/c/cd/IMG_6042.JPG" class="shadow" style="float:left;width:200px;margin:10px"></a></html><br />
<br />
<p>'''Would any of your project ideas raise safety issues in terms of:'''</p><br />
<br />
<p>'''''Researcher Safety,'''''</p><br />
<br />
<p>Nicotiana tabacum - No. NT cells are common cell lines used for decades in plant research. No known hazards have been associated with NT cell research.<br />
<br />
Reporter genes – No. Fluorescent proteins are a staple of molecular biology, and no known hazards have been associated with their use. Our promoters also pose no threat.</p><br />
<br />
<br />
<p>'''''Public Safety,'''''</p><br />
<br />
<p>Nicotiana tabacum - No. We do not intend on developing the project in any way such that the public would encounter our project. Even so, in a hypothetical commercial development, NT cells are not expected to put the public at risk. NT cells are not viable outside a nutrient-rich environment.</p><br />
<br />
<p>None of the parts we intend to make, promoters or fluorescent reporters, have shown any health risks to date.</p><br />
<br />
<br />
<p>'''''Environmental Safety'''''</p><br />
<br />
<p>- No. While other plant models could conceivably cross with wild-type plants and generate unforeseeable hybrids, NT cells mitigate that risk. Because NT cells are incapable of sexual reproduction and can only proliferate through clonal propagation in their nutrient-rich media. Containment of the cells is easier to manage with less risk should the NT cells ever breach containment. Therefore, we would not expect any of our promoters or reporter genes to reach the environment. We are ensuring proper containment of transformed Agrobacterium.</p><br />
<br />
<br />
<p>'''Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''</p> <br />
<br />
<p>- No. Our reporter genes are standard fluorescent proteins used repeatedly not just in iGEM but research abroad. Our modified plasmid poses no risk to the researchers, public, or environment. The inducible promoters selected are found naturally in plants and are predicted to pose no risk.</p><br />
<br />
<br />
<p>'''Is there a local biosafety group, committee, or review board at your institution? <br />
<br />
If yes, what does your local biosafety group think about your project?'''</p><br />
<br />
<p>- Yes. The University of Nevada, Reno Institutional Biosafety Committee supports our project, especially with regard to the fact every member of the iGEM team completed training on NIH Guidelines on Recombinant Organisms. An additional safety course conducted by the University of Nevada, Reno Environmental Health & Safety Department trained each member in the certified Lab Safety Workshop on chemical safety, biological safety, chemical hygiene, ventilation, and waste management. Therefore, all iGEM members have been trained in the handling and disposal of transgenic bacteria.</p> <br />
<br />
<br />
'''Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?''' <br />
<br />
<p>The steps previously and currently taken by iGEM teams to design systems where modified organisms can be screened or terminated under certain conditions are excellent ways of providing safety. Certainly, controlling where and how organisms can grow helps to ensure modified organisms do not unintentionally interact with the environment. These are all internal controls, and while 99.9% reliable, we believe the risk of unforeseen mutation cannot be overlooked. Several systems of control will have a few layers of regulation, but there could likely be one nexus of vulnerability that the whole system hinges upon, and the question must be asked what happens if that node of control fails? A dysfunctional fluorescent protein may not deserve attention, but genes regulating proliferation, reproduction, or repressors could cause bacteria, fungi, or plants to escape containment. Adding layers of regulation do help, but ultimately they affect the quality of the plasmids or genes used and may ultimately hinder the goals set for the project.</p><br />
<br />
<p>Therefore, we propose one additional way of providing safety in an iGEM doomsday scenario to help in the event of some future iGEM bacteria that infects humans or plants, affecting public health or crops. Perhaps, as iGEM grows and commercial applications become apparent, for plant projects, iGEM could require a constitutively expressed fluorescent protein alongside any novel gene to track the plants. iGEM-compatible internal controls have been designed, for example Harvard’s project, but in the rare event those mechanisms fail, a constitutively expressed fluorescent protein would be an easily identifiable way of tracking iGEM plants.</p><br />
<p>&nbsp;</p><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
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!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/CD2InducibleTeam:Nevada/CD2Inducible2010-10-25T05:42:21Z<p>Mpolasko: </p>
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<p>&nbsp;</p><br />
<br />
== Promoters ==<br />
<br />
<br />
<br />
<br />
<br />
<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/DREB1C" tabindex="1">DREB 1C</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/RD29A" tabindex="2">RD29A</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/35S" tabindex="3">35S</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/CD2Inducible" tabindex="4">CD2+ Inducible</a></li><br />
</ul><br />
</div><br />
</html><br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png '''Cadmium Inducible Promoter''' [[Team:Nevada/registry submissions]]<br />
<br />
----<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/8/8f/Hileary.jpg"><img src="https://static.igem.org/mediawiki/2010/8/8f/Hileary.jpg" class="shadow" style="float:left;width:200px;margin:10px"></a><br />
</html><br />
<p>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 and Cramer). 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 (Bovet 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.</p><br />
<p>&nbsp;</p><br />
<p>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><br />
<br><br />
'''References'''<br />
<br>'''Bovet et al.''' Transcript levels of AtMRP3 after cadmium treatment: induction of AtMRP3. Plant, Cell and Environment., 26: 371-381, 2003.<br />
<br>'''Gobe and Cramer.''' Mitochondria, reactive oxygen species and cadmium toxicity in the kidney. Toxicology Letters., 198: 49-55, 2010.<br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/promotersTeam:Nevada/promoters2010-10-25T05:41:21Z<p>Mpolasko: </p>
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<div>{{Nevada_css}}<br />
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<p>&nbsp;</p><br />
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== Promoters ==<br />
<br />
<br />
<br />
<br />
<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/DREB1C" tabindex="1">DREB1C</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/RD29A" tabindex="2">RD29A</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/35S" tabindex="3">35S</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/CD2Inducible" tabindex="4">CD2+ Inducible</a></li><br />
</ul><br />
</div><br />
</html><br />
<br />
<html><br />
<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><br />
</html><p>The Nevada team has created environmental stress sensors by using <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> and <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter">DREB1C</a></html> promoters to express red fluorescent protein and other bio-fluorescent markers. When induced by environmental stress, plants carrying these genes can easily be detected by the farmer walking through his field or by a plane flying over acres of farmland. Promoter elements are short and easy to work with. They also allow for modification and specialization. <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter">DREB1C</a></html> and <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> both have multiple binding motifs in their promoter regions allowing for variation in expression levels and the particular stresses that induce them. 35s is a constitutive promoter that can be valuable for control groups in stress and other plant response research.</p><br />
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<!--- The Mission, Experiments ---><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/ccdBTeam:Nevada/ccdB2010-10-25T05:39:29Z<p>Mpolasko: </p>
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== ccdB ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
'''ccdB minimal gene''' [[Team:Nevada/registry submissions]]<br />
<br />
----<br />
<html><a href="https://static.igem.org/mediawiki/2010/a/a0/Randy_and_Vadim.jpg"><img src="https://static.igem.org/mediawiki/2010/a/a0/Randy_and_Vadim.jpg" class="shadow" style="float:left;width:400px;margin:10px"></a></html><p>The ccdB gene is a known topoisomerase II poison and will kill most commercially available E. coli cell lines. The ccdB gene can be used as a selectable marker by placing it in the multicloning region of a plasmid and propagating it in ccdB resistant cell lines (e.g. DB3.1 from New England Biolabs). During cloning, the ccdB gene can be switched out for your gene of interest and transformed into a cell line that is susceptible to the toxic effects of ccdB (e.g NEBβ cells from New England Biolabs). Colonies that grow on plates should contain the plasmid and your gene of interest. If the ccdB gene is still present in the plasmid, the plasmid will kill any colonies where it is still present. This is an improvement to the current ccdB cell death gene part (BBa_P1010) as it does not contain the inactive ccdA gene or an unknown stuffer region.</p><br />
<p>&nbsp;</p><br />
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<p>'''Our Method:''' ccdB was amplified using Polymerase Chain Reaction (PCR) with primers designed to contain the specific BioBrick prefix and suffix. The amplified fragment was then TOPO-cloned into a PCR-Blunt II vector (Invitrogen). The vector was then cut with EcoRI and PstI restriction enzymes and the ccdB fragment was transformed into the iGEM compatible vector pSB1C3.<html><a href="https://static.igem.org/mediawiki/2010/0/05/CcdB_method.png"><img src="https://static.igem.org/mediawiki/2010/0/05/CcdB_method.png" class="shadow" style="float:center;width:850px;margin:10px"></a></html></p><br />
<p>&nbsp;</p><br />
----<br />
<br />
<p>'''Our Results:'''<br />
<br><br />
*ccdB in the PCR-Blunt II vector were transformed in three different cell lines: NEB10β cells (New England Biolabs), Omni Max 2 cells (Invitrogen), and DB3.1 cells (New England Biolabs). NEB10β and Omni Max 2 cell lines are not resistant to the toxic properties of ccdB. No colonies were present in those two cell lines but were present in DB3.1 cell lines (Right). <br />
<br><br />
*PCR gel confirming the presence of ccdB in amplified sample(Left). <html><a href="https://static.igem.org/mediawiki/2010/6/63/Ccdb_results.png"><img src="https://static.igem.org/mediawiki/2010/6/63/Ccdb_results.png" class="shadow" style="float:center;width:850px;margin:10px"></a></html></p><br />
<p>&nbsp;</p><br />
<br />
----<br />
<br />
<p>'''Vectors Used:'''</p><html><a href="https://static.igem.org/mediawiki/2010/f/f4/Vectors_ccdB.png"><img src="https://static.igem.org/mediawiki/2010/f/f4/Vectors_ccdB.png" class="shadow" style="float:left;width:500px;margin:10px"></a></html><br />
<p>&nbsp;</p><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/TransformationsTeam:Nevada/Transformations2010-10-25T05:37:01Z<p>Mpolasko: </p>
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== NT Cell Transformations ==<br />
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<html><a href="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG"><img src="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG" class="shadow" style="float:left;width:500px;margin:10px"></a><br />
</html><br />
<br />
<br />
<p><span style="font-size:18px;font-weight:bold;text-decoration:underline">NT Cell Protocol</span></p><br />
<br />
<p>'''CARE OF A NT CULTURE'''</p><br />
(adapted from a letter by Dr. Michael Sullivan) <br />
To readapt a culture on plates, simply transfer some of the cells back into liquid <br />
media. We usually pipette the cell suspension up and down to break up any clumps. It <br />
may be best to start out with a smaller culture volume when you first go back to liquid; <br />
BY-2 seems to be a bit happier if it isn't seeded to thinly. Allow the culture to grow <br />
until it is the consistency of thin applesauce. At this point, you should be able to go to <br />
a 50 ml culture and start subculturing as described below. In our experience, wild-type <br />
BY-2 readapts quickly to liquid to give a smooth culture; transformed lines tend to be more <br />
variable, with some producing smooth cultures, some producing clumpy ones, and some going <br />
back and forth between these two states. We've found that clumpy cultures do not interfere <br />
with our half-live measurements, although manipulating them can be a bit more difficult. <br />
<br />
We grow our liquid cultures in 50 ml of media in 250 ml baffle flasks at 28 degrees C <br />
with gentle shaking (150 rpm). Using a baffle flask does not seem to be a requirement, many <br />
people grow these cells in regular flasks with no problem. We subculture them once a week <br />
by transferring 5% of the culture to fresh media. We generally maintain two flasks <br />
(in two separate shakers) of two separate subcultures (one subcultured Monday, one on Friday) <br />
in case one of the cultures crashes. Also, you can maintain the culture on a plate. <br />
Note that, for liquid cultures of transformed lines, we usually use a smaller culture volume, <br />
e.g. 10 ml, for convenience. <br />
<br />
<br />
'''NT KC MEDIA - LIQUID OR PLATES<br />
NT LIQUID:''' <br />
(amounts are for 1L of media): <br />
750 ml H2O <br />
4.3 g MS salts (add slowly to liquid) <br />
30 g Sucrose <br />
50 ml 20X MES pH 5.7 <br />
10 ml B1 -inositol <br />
3 ml Miller's I <br />
10 ml 2,4-D, 10-4 M <br />
pH to 5.7 with 0.1 N KOH <br />
Bring volume to 1000ml <br />
Autoclave <br />
<br />
'''SOLID MEDIA:''' <br />
For plates only: Add to flasks 7 g/ L Phytagar before autoclaving <br />
Add kanamycin (100 mg/ml) <br />
Add carbenicillin (250 mg/ml) <br />
<br />
'''MEDIA COMPONENTS:''' <br />
Miller's I: 60 g KH2 PO4 per liter <br />
20 X MES: 10 g MES per liter, pH to 5.7 with 1N KOH <br />
B1 - Inositol: 0.1 g Thiamine, 10.0 g myo-inositol, H2O to final volume of 1 liter <br />
<br />
<br />
<br />
<p><span style="font-size:18px;font-weight:bold;text-decoration:underline">Transformation Protocol</span></p><br />
<p>'''NT Cell Transformation with Agrobactrium'''</p><br />
<p>'''Day 1:''' </p><br />
<p>1. Grow up 1 ml of the Agrobacterium overnight in LB + all selective drugs. This culture may be started from frozen glycerol cultures if necessary. </p><br />
<br />
<p>'''Day 2:''' </p><br />
<p>2. NT cells are used 3 days after splitting the NT cell culture. 4 ml of NT cells are required for each transformation with an additional 4 ml for the control culture which receives no bacteria. </p><br />
<br />
3. 1 ul Acetosyringone (20 mM in ethanol) is added per ml of NT cells. Typically treat the whole 50 ml culture at this point and discard any that is left over when I'm finished. <br />
<br />
4. Using a 10 ml pipette, the NT cells are pipetted in and out about 20 times. This helps to induce small lesions in the cells and increases the efficiency of the transformation. <br />
<br />
5. 75 ul of bacteria (dense growth) or 100 ul (moderate growth) are added to a petri dish containing 4 ml of NT cells (from step 4) and mixed thoroughly. REMEMBER TO INCLUDE A CONTROL HAVING NO BACTERIA. <br />
<br />
6. Wrap plates with parafilm and incubate for 3 days at 28 degrees C. <br />
<br />
<p>'''Day 5:''' </p><br />
7. To each plate add 10 ml of NT liquid medium containing 500 (u)g/ml carbenicillin (NTC).<br />
<br />
8. Collect cells off of the plates into 50 ml conical tubes and fill with NTC. <br />
<br />
9. Centrifuge at 1K for 4 minutes at room temperature in a swinging bucket rotor. <br />
<br />
10. Aspirate off liquid using pipet capped with a sterile blue 1 ml top. Change tip for each sample.<br />
<br />
11. Resuspend in 50 ml NTC and repeat spin. <br />
<br />
12. Repeat step 10 and 11 - 1 or 2 times. Strains that are fairly sensitive to carb require fewer washes. <br />
<br />
13. Resuspend cells in approximately 5 ml NTC and plate 2.0 ml on 2 NTKC (kanamycin 100 ug/ml carbenicillin 500 ul/ml) plates. When there is no longer lots of fluid on the plates (leave the plates open in the hood a few minutes of necessary), wrap them in parafilm.<br />
<br />
14. Incubate at 28 degrees C. Transformants should be large enough to transfer to fresh NTKC plates in 3-4 weeks. <br />
<br />
'''Supplies for each transformation (Remember the controls):''' <br />
<br />
Day 1 Supplies: <br />
LB + appropriate drugs<br />
Agrobacterium containing plasmid for transformation <br />
<br />
Day 2 Supplies: <br />
1 ml Agrobacterium overnight culture <br />
4 ml BY-2 cells - 3 days post subculture <br />
4 ul acetosyringone (20 mM) found in the (-20) refrigerator freezer <br />
pipets and pipetman <br />
1 petri plate <br />
<br />
Day 5 Supplies: <br />
200 ml NTC liquid <br />
50 ml conical tube <br />
swinging bucket centrifuge at room temp aspiration setup with 5 ml pipet capped with 1 ml blue tip <br />
2 NTKC plates <br />
pipetmen and tips <br />
<br />
<br />
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<html><a href="https://static.igem.org/mediawiki/2010/4/4f/IMG_6028.JPG"><img src="https://static.igem.org/mediawiki/2010/4/4f/IMG_6028.JPG" class="shadow" style="float:left;width:430px;margin:10px"></a><br />
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<html><a href="https://static.igem.org/mediawiki/2010/3/3d/IMG_6035.JPG"><img src="https://static.igem.org/mediawiki/2010/3/3d/IMG_6035.JPG" class="shadow" style="float:right;width:430px;margin:10px"></a></html><br />
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<p>'''Left:''' NT Cell Culture after about 5 days. '''Right:''' NT Cell Transformation with RD29A (Cold, Drought, Salt) Inducible Promoter.</p><br />
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<!--- The Mission, Experiments ---><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/CompositeTeam:Nevada/Composite2010-10-25T05:29:53Z<p>Mpolasko: </p>
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== Composite Parts ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*RD29A Promoter + Strong Plant Kozak (RBS) + RFP [[Team:Nevada/registry submissions]]<br />
*35S Promoter + (Strong Plant RBS + GFP) From K414001 [[Team:Nevada/registry submissions]]<br />
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<html><br />
<a href="https://static.igem.org/mediawiki/2010/6/60/Composite_RD29A.png"><img src="https://static.igem.org/mediawiki/2010/6/60/Composite_RD29A.png" class="shadow" style="float:left;width:500px;margin:10px"></a><br />
</html><br />
<p><span style="text-decoration:underline;">RD29A –RFP</span>: This composite is designed so that transformed plants will yield red fluorescent protein under cold, drought, or high soil salinity conditions. </p><br />
<br />
<p><span style="text-decoration:underline;">35S – GFP</span>: This composite is designed so that transformed plants will constitutively express green fluorescent protein. Having this constant expression provides the basis to measure any other inducible fluorescent proteins’ fluorescents relative to an internal control. </p><br />
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<!--- The Mission, Experiments ---><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
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!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/TransformationsTeam:Nevada/Transformations2010-10-25T05:29:16Z<p>Mpolasko: </p>
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== NT Cell Transformations ==<br />
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<p>Insert Text <html><a href="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG"><img src="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG" class="shadow" style="float:left;width:500px;margin:10px"></a><br />
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'''Protocol'''<br />
<br />
'''CARE OF A NT CULTURE'''<br />
(adapted from a letter by Dr. Michael Sullivan) <br />
To readapt a culture on plates, simply transfer some of the cells back into liquid <br />
media. We usually pipette the cell suspension up and down to break up any clumps. It <br />
may be best to start out with a smaller culture volume when you first go back to liquid; <br />
BY-2 seems to be a bit happier if it isn't seeded to thinly. Allow the culture to grow <br />
until it is the consistency of thin applesauce. At this point, you should be able to go to <br />
a 50 ml culture and start subculturing as described below. In our experience, wild-type <br />
BY-2 readapts quickly to liquid to give a smooth culture; transformed lines tend to be more <br />
variable, with some producing smooth cultures, some producing clumpy ones, and some going <br />
back and forth between these two states. We've found that clumpy cultures do not interfere <br />
with our half-live measurements, although manipulating them can be a bit more difficult. <br />
<br />
We grow our liquid cultures in 50 ml of media in 250 ml baffle flasks at 28 degrees C <br />
with gentle shaking (150 rpm). Using a baffle flask does not seem to be a requirement, many <br />
people grow these cells in regular flasks with no problem. We subculture them once a week <br />
by transferring 5% of the culture to fresh media. We generally maintain two flasks <br />
(in two separate shakers) of two separate subcultures (one subcultured Monday, one on Friday) <br />
in case one of the cultures crashes. Also, you can maintain the culture on a plate. <br />
Note that, for liquid cultures of transformed lines, we usually use a smaller culture volume, <br />
e.g. 10 ml, for convenience. <br />
<br />
<br />
'''NT KC MEDIA - LIQUID OR PLATES<br />
NT LIQUID:''' <br />
(amounts are for 1L of media): <br />
750 ml H2O <br />
4.3 g MS salts (add slowly to liquid) <br />
30 g Sucrose <br />
50 ml 20X MES pH 5.7 <br />
10 ml B1 -inositol <br />
3 ml Miller's I <br />
10 ml 2,4-D, 10-4 M <br />
pH to 5.7 with 0.1 N KOH <br />
Bring volume to 1000ml <br />
Autoclave <br />
<br />
'''SOLID MEDIA:''' <br />
For plates only: Add to flasks 7 g/ L Phytagar before autoclaving <br />
Add kanamycin (100 mg/ml) <br />
Add carbenicillin (250 mg/ml) <br />
<br />
'''MEDIA COMPONENTS:''' <br />
Miller's I: 60 g KH2 PO4 per liter <br />
20 X MES: 10 g MES per liter, pH to 5.7 with 1N KOH <br />
B1 - Inositol: 0.1 g Thiamine, 10.0 g myo-inositol, H2O to final volume of 1 liter <br />
<br />
<br />
'''TRANSFORMATION PROTOCOL:<br />
NT Cell Transformation with Agrobactrium''' <br />
'''Day 1:''' <br />
1. Grow up 1 ml of the Agrobacterium overnight in LB + all selective drugs. This culture may be started from frozen glycerol cultures if necessary. <br />
<br />
'''Day 2:''' <br />
2. NT cells are used 3 days after splitting the NT cell culture. 4 ml of NT cells are required for each transformation with an additional 4 ml for the control culture which receives no bacteria. <br />
<br />
3. 1 ul Acetosyringone (20 mM in ethanol) is added per ml of NT cells. Typically treat the whole 50 ml culture at this point and discard any that is left over when I'm finished. <br />
<br />
4. Using a 10 ml pipette, the NT cells are pipetted in and out about 20 times. This helps to induce small lesions in the cells and increases the efficiency of the transformation. <br />
<br />
5. 75 ul of bacteria (dense growth) or 100 ul (moderate growth) are added to a petri dish containing 4 ml of NT cells (from step 4) and mixed thoroughly. REMEMBER TO INCLUDE A CONTROL HAVING NO BACTERIA. <br />
<br />
6. Wrap plates with parafilm and incubate for 3 days at 28 degrees C. <br />
<br />
'''Day 5:''' <br />
7. To each plate add 10 ml of NT liquid medium containing 500 (u)g/ml carbenicillin (NTC).<br />
<br />
8. Collect cells off of the plates into 50 ml conical tubes and fill with NTC. <br />
<br />
9. Centrifuge at 1K for 4 minutes at room temperature in a swinging bucket rotor. <br />
<br />
10. Aspirate off liquid using pipet capped with a sterile blue 1 ml top. Change tip for each sample.<br />
<br />
11. Resuspend in 50 ml NTC and repeat spin. <br />
<br />
12. Repeat step 10 and 11 - 1 or 2 times. Strains that are fairly sensitive to carb require fewer washes. <br />
<br />
13. Resuspend cells in approximately 5 ml NTC and plate 2.0 ml on 2 NTKC (kanamycin 100 ug/ml carbenicillin 500 ul/ml) plates. When there is no longer lots of fluid on the plates (leave the plates open in the hood a few minutes of necessary), wrap them in parafilm.<br />
<br />
14. Incubate at 28 degrees C. Transformants should be large enough to transfer to fresh NTKC plates in 3-4 weeks. <br />
<br />
'''Supplies for each transformation (Remember the controls):''' <br />
<br />
Day 1 Supplies: <br />
LB + appropriate drugs<br />
Agrobacterium containing plasmid for transformation <br />
<br />
Day 2 Supplies: <br />
1 ml Agrobacterium overnight culture <br />
4 ml BY-2 cells - 3 days post subculture <br />
4 ul acetosyringone (20 mM) found in the (-20) refrigerator freezer <br />
pipets and pipetman <br />
1 petri plate <br />
<br />
Day 5 Supplies: <br />
200 ml NTC liquid <br />
50 ml conical tube <br />
swinging bucket centrifuge at room temp aspiration setup with 5 ml pipet capped with 1 ml blue tip <br />
2 NTKC plates <br />
pipetmen and tips <br />
<br />
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<html><a href="https://static.igem.org/mediawiki/2010/4/4f/IMG_6028.JPG"><img src="https://static.igem.org/mediawiki/2010/4/4f/IMG_6028.JPG" class="shadow" style="float:left;width:430px;margin:10px"></a><br />
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<p>'''Left:''' NT Cell Culture after about 5 days. '''Right:''' NT Cell Transformation with RD29A (Cold, Drought, Salt) Inducible Promoter.</p><br />
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<!--- The Mission, Experiments ---><br />
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!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/promotersTeam:Nevada/promoters2010-10-25T05:26:47Z<p>Mpolasko: </p>
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== Promoters ==<br />
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<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/DREB1C" tabindex="1">DREB1C</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/RD29A" tabindex="2">RD29A</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/35S" tabindex="3">35S</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/CD2Inducible" tabindex="4">CD2+ Inducible</a></li><br />
</ul><br />
</div><br />
</html><br />
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<html><br />
<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><br />
</html><p>The Nevada team has created environmental stress sensors by using <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> and <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter">DREB1C</a></html> promoters to express red fluorescent protein and other bio-fluorescent markers. When induced by environmental stress, plants carrying these genes can easily be detected by the farmer walking through his field or by a plane flying over acres of farmland. Promoter elements are short and easy to work with. They also allow for modification and specialization. <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter">DREB1C</a></html> and <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> both have multiple binding motifs in their promoter regions allowing for variation in expression levels and the particular stresses that induce them. 35s is a constitutive promoter that can be valuable for control groups in stress and other plant response research.</p><br />
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|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/plant_compatible_reportersTeam:Nevada/plant compatible reporters2010-10-25T05:23:47Z<p>Mpolasko: </p>
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<p>&nbsp;</p><br />
== Reporters ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*Strong Plant RBS (Kozak sequence) + GFP from E0040 [[Team:Nevada/registry submissions]]<br />
*Strong Plant RBS (Kozak sequence) + EYFP from E0030 [[Team:Nevada/registry submissions]]<br />
*Strong Plant RBS (Kozak sequence) + mCherry from J06504 [[Team:Nevada/registry submissions]]<br />
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<p style="text-align:center;"><span style="text-decoration:underline;font-weight:bold">Do You Have A Green Thumb?</span></p><br />
<p>The Nevada iGEM team along with a handful of other teams this year are looking to expand iGEM to the world of plants. iGEM will finally have a medium to explore eukaryotic gene manipulation without requiring yeast farms or human experimentation. Plants provide a kingdom of exploration that could make iGEM a potential attractant to commercial biotech firms as it brings the possibilities of synthetic biology out into the open from the patents and trade secret shadows which a lot of firms rely on today.</p> <br />
<br />
<p>Engaging a whole new realm of organisms requires fundamental understanding of those organisms and how they differ from the current models. That understanding from iGEM teams is essential to its success. Obviously, plants come with their own unique promoters, activators, and repressors with which to play. We added a few plant promoters to the iGEM registry for future teams to use. Also, there will need to be new consideration as to what types of proteins with which we can transform the plants. Fortunately, some essential eukaryotic proteins that iGEM has dealt with, like the fluorescent proteins, have already been used in plant experimentation.</p><br />
<br />
As far as testing new models, we believe our NT cells may provide a cheaper, faster alternative to teams interested in experimenting with plants. Also, the NT cells are safer to use and pose less risk to the environment then experimenting with actual plants. <br />
<br />
<p style="text-align:center;"><span style="text-decoration:underline;font-weight:bold">Building Consensus</span></p><br />
<br />
While all the aforementioned issues are important, the aspect of plant engineering that we believe is fundamental for future iGEM teams to consider is the ribosome binding site (RBS). RBS can differ between species, but it varies widely between eukaryotes, such as yeast, animals, and plants. The term RBS can be misleading because ribosomes can weakly associate with RNA as it “scans” along the sequence. Why is the ribosome “scanning?” Ribosomes initiate translation, and the “start” site that we have all been taught for eukaryotes is the methionine sequence AUG (or ATG if you are biased towards DNA). However, almost thirty years ago, a researcher named Kozak discovered that it is not simply AUG which initiates translation but the context of that AUG, the surrounding sequence, influenced whether translation actually began with one AUG sequence versus another. These context sequences, as they have been discovered in different organisms, eventually have been named Kozak sequences. <br />
<br />
Even among plants, there can be different Kozak sequences. Where we decided to contribute to iGEM was to supply the registry with the first fluorescent proteins with plant compatible RBS or Kozak sequences. We have chosen a generically ‘strong’ Kozak sequence that should provide the maximum translational efficiency for dicots, but it should also work generally well enough in most if not all plants. Our sequence is AAA AAA AAA ACA upstream of the AUG. An important aspect of Kozak sequences one should consider is there are both an upstream component and a downstream component. The string of purines upstream is associated with many plant Kozak sequences, but almost equally important is to have a G at the +4 position, or immediately following the AUG. Therefore, AAAAAAAAACA'''AUG'''G is likely to have the highest translational efficiency. Fortunately, two of the florescent proteins, EYFP and mCherry have this context. GFP, however does not. It is missing the G at +4 which will hurt its translational efficiency. Instead, a C occupies that position which codes for arginine, R. There is no codon for arginine that starts with G. Unless a known mutation can be made, we may stuck with that hindrance. However, we have attempted to compensate in one of our composite parts, 35S GFP. 35S is a constitutive plant promoter. Ideally, the high transcriptional activity can compensate for the weakened translational efficiency. <br />
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<p style="text-align:center;"><span style="text-decoration:underline;font-weight:bold">Engineering Possibilities (Fine tuning your translation)</span></p><br />
As we see it, there are three ways future iGEM teams could engage the plant Kozak sequence to modify gene expression in plants: identity, distance, or deking.<br />
<br />
'''1)Identity''': The most obvious way of affecting translational efficiency would be to alter the Kozak sequences. Having genes each prefaced with the same promoter but with different Kozak contextual sequences would tier the levels of expression. One could have an optimum Kozak like the one we have submitted and also engineer a weaker Kozak sequence for another gene which has relatively 50% expression compared to the optimum gene expressed. Consulting literature or experimenting in less-frequently researched plants will allow for greater variability in controlling expression. <br />
<br />
'''2)Distance''': Another way to affect your protein expression would be how far the ‘true’ Kozak sequence is relative to the 5’ cap. A strong Kozak sequence means nothing if it is several hundred base pairs away from the end of the promoter. Because our team wanted to supply genes with maximal expression, our parts are intended to be placed immediately behind the promoter. Yet, engineering plasmids that put gaps between the promoter and actual Kozak, or primers designed to put more space in between the promoter and start site, could also be one way of dialing the levels of expression. <br />
<br />
'''3)Deking''':(Realizing a team from a desert is using a hockey term): The “fake out.” A third alternative that combines the principles of identity and distance is to create one, two, or a few pseudo-start sites. Psuedo-start sites means one would engineer AUG sequences upstream of the actual, desired one. These sequences would be in a poorer context and/or would translate into little nonsense peptides that theoretically have no function. Think of them as siAUG (short interfering AUG sites). These fake sites would knockdown expression. <br />
<br />
In Summary, Kozak sequences have plenty of promise in the engineering side of iGEM, but Kozak sequences are also a necessity that all iGEM teams must consider if they are to express proteins in plants.<br />
<br />
<span style="text-decoration:underline;font-weight:bold">References</span><br />
<br />
'''Agarwal, S., Jha, S., Sanyal, I., Amla, D.V.''' (2009) Effect of point mutations in translation initiation context on the expression of recombinant human alpha1-proteinase inhibitor in transgenic tomato plants. ''Plant Cell Reports''. 28: 1791-1798.<br />
<br />
'''Joshi, C.P., Zhou, H., Huang, X., Chiang, V.L.''' (1997) Context sequences of translation initiation codon in plants. ''Plant Molecular Biology''. 35: 993-1001.<br />
<br />
'''Kozak, M.''' (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribososomes. ''Cell''. 44: 283-92.<br />
<br />
'''Matsuda, D., Dreher, T.W.''' (2006) Close spacing of AUG initiation codons confers dicistronic character on a eukaryotic mRNA. ''RNA''. 12: 1138-1349.<br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/CompositeTeam:Nevada/Composite2010-10-25T05:14:14Z<p>Mpolasko: </p>
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<p>&nbsp;</p><br />
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== Composite Parts ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*RD29A Promoter + Strong Plant Kozak (RBS) + RFP [[Team:Nevada/registry submissions]]<br />
*35S Promoter + (Strong Plant RBS + GFP) From K414001 [[Team:Nevada/registry submissions]]<br />
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<html><br />
<a href="https://static.igem.org/mediawiki/2010/6/60/Composite_RD29A.png"><img src="https://static.igem.org/mediawiki/2010/6/60/Composite_RD29A.png" class="shadow" style="float:left;width:500px;margin:10px"></a><br />
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<p><span style="text-decoration:underline;">RD29A –RFP</span>: This composite is designed so that transformed plants will yield red fluorescent protein under cold conditions. </p><br />
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<p><span style="text-decoration:underline;">35S – GFP</span>: This composite is designed so that transformed plants will constitutively express green fluorescent protein. Having this constant expression provides the basis to measure any other inducible fluorescent proteins’ fluorescents relative to an internal control. </p><br />
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!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/TeamTeam:Nevada/Team2010-10-25T04:59:57Z<p>Mpolasko: </p>
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<p style="text-align:left;">&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Meet the Team</span><br />
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<p style="text-align:center;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Advisors</span><br />
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[[Image:Christie.jpg|center|thumb|alt=alt text|<p style="text-align:center;"><span style="font-family:Times;font-size:12pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Dr. Christie Howard</span></p>]]<br />
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[[Image:Shintani.jpg|center|thumb|alt=alt text|<p style="text-align:center;"><span style="font-family:Times;font-size:12pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Dr. David Shintani</span></p>]]<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Undergraduates</span><br />
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<gallery widths=170px heights=170px perrow=4><br />
Image:Hilary UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span></p><br />
Image:mpolasko2.JPG|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span></p><br />
Image:Randy.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span></p><br />
Image:Copley.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span></p><br />
Image:Bryson UNR 2.png| <p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span></p><br />
Image:Gladwill.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span></p><br />
Image:Image-Elaine.jpeg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span></p><br />
Image:Sam UNR .png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span></p><br />
&nbsp;<br />
Image:Nick UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span></p><br />
Image:Hileary.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span></p><br />
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<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:normal;text-decoration:bold;color:#15317E;background-color:ffffff;">What We Did</span><br />
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----<br />
<br />
<div style="padding:10px;clear:both"><br />
{| border="0" cellpadding="0" cellspacing="20"<br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Team President</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">NT Cell Transformations</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter + Plant Kozak + RFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + EYFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + mCherry</span><br />
*<span style="font-size:12pt;font-weight:bold;">Composite - 35s Promoter + Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;"> DREB1C Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">35S Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Modeling</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">RD29A Promoter</span><br />
|-<br />
|&nbsp;<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span><br />
|-<br />
|&nbsp;<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|}<br />
----<br />
<br />
</div><br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/TeamTeam:Nevada/Team2010-10-25T04:47:35Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Picture Team.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
<p style="text-align:left;">&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Meet the Team</span><br />
</p><br />
----<br />
<div style="padding: 10px 0 15px 15px;"><br />
<br />
<html><br />
<a href="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG" class="shadow" style="float:left;margin:0px;width:600px"></a><br />
</html><br />
<br />
<p style="text-align:center;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Advisors</span><br />
</p><br />
----<br />
<br />
[[Image:Christie.jpg|center|thumb|alt=alt text|Dr. Christie Howard]]<br />
<br />
[[Image:Shintani.jpg|center|thumb|alt=alt text|Dr. Dave Shintani]]<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Undergraduates</span><br />
</p><br />
----<br />
<gallery widths=170px heights=170px perrow=4><br />
Image:Hilary UNR.png|Hilary Allen<br />
Image:mpolasko2.JPG|Matthew Polasko<br />
Image:Randy.jpg|Randy Pares<br />
Image:Copley.jpg|Christian Copley<br />
Image:Bryson UNR 2.png|Bryson Wheeler<br />
Image:Gladwill.jpg|Vadim Gladwill<br />
Image:Image-Elaine.jpeg|Elaine Bersaba<br />
Image:Sam UNR .png|Samantha Lee<br />
&nbsp;<br />
Image:Nick UNR.png|Nick Noel<br />
Image:Hileary.jpg|Richard Hilleary<br />
</gallery><br />
<br />
<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:normal;text-decoration:bold;color:#15317E;background-color:ffffff;">What We Did</span><br />
</p><br />
----<br />
<br />
<div style="padding:10px;clear:both"><br />
{| border="0" cellpadding="0" cellspacing="20"<br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Team President</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">NT Cell Transformations</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter + Plant Kozak + RFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + EYFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + mCherry</span><br />
*<span style="font-size:12pt;font-weight:bold;">Composite - 35s Promoter + Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;"> DREB1C Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">35S Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Modeling</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">RD29A Promoter</span><br />
|-<br />
|&nbsp;<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span><br />
|-<br />
|&nbsp;<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|}<br />
----<br />
<br />
</div><br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/TeamTeam:Nevada/Team2010-10-25T04:45:20Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Picture Team.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
<p style="text-align:left;">&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Meet the Team</span><br />
</p><br />
----<br />
<div style="padding: 10px 0 15px 15px;"><br />
<br />
<html><br />
<a href="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG" class="shadow" style="float:left;margin:0px;width:600px"></a><br />
</html><br />
<br />
<p style="text-align:center;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Advisors</span><br />
</p><br />
----<br />
<br />
[[Image:Christie.jpg|center|thumb|alt=alt text|Dr. Christie Howard]]<br />
<br />
[[Image:Shintani.jpg|center|thumb|alt=alt text|Dr. Dave Shintani]]<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Undergraduates</span><br />
</p><br />
----<br />
<gallery widths=170px heights=170px perrow=4><br />
Image:Hilary UNR.png|Hilary Allen<br />
Image:mpolasko2.JPG|Matthew Polasko<br />
Image:Randy.jpg|Randy Pares<br />
Image:Copley.jpg|Christian Copley<br />
Image:Bryson UNR 2.png|Bryson Wheeler<br />
Image:Gladwill.jpg|Vadim Gladwill<br />
Image:Image-Elaine.jpeg|Elaine Bersaba<br />
Image:Sam UNR .png|Samantha Lee<br />
&nbsp;<br />
Image:Nick UNR.png|Nick Noel<br />
Image:Hileary.jpg|Richard Hilleary<br />
</gallery><br />
<br />
<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:normal;text-decoration:bold;color:#15317E;background-color:ffffff;">What We Did</span><br />
</p><br />
----<br />
<br />
<div style="padding:10px;clear:both"><br />
{| border="0" cellpadding="0" cellspacing="20"<br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Team President</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">NT Cell Transformations</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter + Plant Kozak + RFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + EYFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + mCherry</span><br />
*<span style="font-size:12pt;font-weight:bold;">Composite - 35s Promoter + Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;"> DREB1C Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">35S Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Modeling</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">RD29A Promoter</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|}<br />
----<br />
<br />
</div><br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/TeamTeam:Nevada/Team2010-10-25T04:40:13Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Picture Team.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
<p style="text-align:left;">&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Meet the Team</span><br />
</p><br />
----<br />
<div style="padding: 10px 0 15px 15px;"><br />
<br />
<html><br />
<a href="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG" class="shadow" style="float:left;margin:0px;width:600px"></a><br />
</html><br />
<br />
<p style="text-align:center;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Advisors</span><br />
</p><br />
----<br />
<br />
[[Image:Christie.jpg|center|thumb|alt=alt text|Dr. Christie Howard]]<br />
<br />
[[Image:Shintani.jpg|center|thumb|alt=alt text|Dr. Dave Shintani]]<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Undergraduates</span><br />
</p><br />
----<br />
<gallery widths=170px heights=170px perrow=4><br />
Image:Hilary UNR.png|Hilary Allen<br />
Image:mpolasko2.JPG|Matthew Polasko<br />
Image:Randy.jpg|Randy Pares<br />
Image:Copley.jpg|Christian Copley<br />
Image:Bryson UNR 2.png|Bryson Wheeler<br />
Image:Gladwill.jpg|Vadim Gladwill<br />
Image:Image-Elaine.jpeg|Elaine Bersaba<br />
Image:Sam UNR .png|Samantha Lee<br />
Image:Nick UNR.png|Nick Noel<br />
Image:Hileary.jpg|Richard Hilleary<br />
</gallery><br />
<br />
<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:normal;text-decoration:bold;color:#15317E;background-color:ffffff;">What We Did</span><br />
</p><br />
----<br />
<br />
<div style="padding:10px;clear:both"><br />
{| border="0" cellpadding="0" cellspacing="20"<br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Team President</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">NT Cell Transformations</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter + Plant Kozak + RFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + EYFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + mCherry</span><br />
*<span style="font-size:12pt;font-weight:bold;">Composite - 35s Promoter + Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;"> DREB1C Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">35S Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Modeling</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">RD29A Promoter</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|}<br />
----<br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-25T04:39:00Z<p>Mpolasko: </p>
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<div>{{Nevada_css}}<br />
[[Image:UNR Notebook.png|border|left|950px]]<br />
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{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
</p><br />
----<br />
==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF<br />
** Analysis of sequence of GFP (PSB1C3)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product - Bands showed at 500 bp<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
** Bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Topocloned PCR product of RD29A<br />
** Streaked single colonies of RD29A (Topo Vector)<br />
** Cell cultured single colonies of RD29A (Topo Vector)<br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
<br />
'''Week of October 24-30:'''</div>Mpolaskohttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-25T04:34:48Z<p>Mpolasko: </p>
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<div>{{Nevada_css}}<br />
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<p>&nbsp;</p><br />
=='''''Team Nevada Notebook'''''==<br />
<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF<br />
** Analysis of sequence of GFP (PSB1C3)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product - Bands showed at 500 bp<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
** Bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Topocloned PCR product of RD29A<br />
** Streaked single colonies of RD29A (Topo Vector)<br />
** Cell cultured single colonies of RD29A (Topo Vector)<br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
<br />
'''Week of October 24-30:'''</div>Mpolaskohttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-25T04:34:25Z<p>Mpolasko: </p>
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[[Image:UNR Notebook.png|border|left|950px]]<br />
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<p>&nbsp;</p><br />
=='''''Team Nevada Notebook'''''==<br />
<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==May==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF<br />
** Analysis of sequence of GFP (PSB1C3)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product - Bands showed at 500 bp<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
** Bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Topocloned PCR product of RD29A<br />
** Streaked single colonies of RD29A (Topo Vector)<br />
** Cell cultured single colonies of RD29A (Topo Vector)<br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
<br />
'''Week of October 24-30:'''</div>Mpolaskohttp://2010.igem.org/Team:Nevada/TeamTeam:Nevada/Team2010-10-25T04:29:31Z<p>Mpolasko: </p>
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<div>{{Nevada_css}}<br />
[[Image:Picture Team.png|border|left|950px]]<br />
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{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
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<br />
<p style="text-align:left;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Meet the Team</span><br />
</p><br />
----<br />
<div style="padding: 10px 0 15px 15px;"><br />
<br />
<html><br />
<a href="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG" class="shadow" style="float:left;margin:0px;width:600px"></a><br />
</html><br />
<br />
<p style="text-align:center;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Advisors</span><br />
</p><br />
----<br />
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[[Image:Christie.jpg|center|thumb|alt=alt text|Dr. Christie Howard]]<br />
<br />
[[Image:Shintani.jpg|center|thumb|alt=alt text|Dr. Dave Shintani]]<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Undergraduates</span><br />
</p><br />
----<br />
<gallery widths=170px heights=170px perrow=4><br />
Image:Hilary UNR.png|Hilary Allen<br />
Image:mpolasko2.JPG|Matthew Polasko<br />
Image:Randy.jpg|Randy Pares<br />
Image:Copley.jpg|Christian Copley<br />
Image:Bryson UNR 2.png|Bryson Wheeler<br />
Image:Gladwill.jpg|Vadim Gladwill<br />
Image:Image-Elaine.jpeg|Elaine Bersaba<br />
Image:Sam UNR .png|Samantha Lee<br />
Image:Nick UNR.png|Nick Noel<br />
Image:Hileary.jpg|Richard Hilleary<br />
</gallery><br />
<br />
<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:normal;text-decoration:bold;color:#15317E;background-color:ffffff;">What We Did</span><br />
</p><br />
----<br />
<br />
<div style="padding:10px;clear:both"><br />
{| border="0" cellpadding="0" cellspacing="20"<br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Team President</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">NT Cell Transformations</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter + Plant Kozak + RFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + EYFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + mCherry</span><br />
*<span style="font-size:12pt;font-weight:bold;">Composite - 35s Promoter + Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;"> DREB1C Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">35S Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Modeling</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">RD29A Promoter</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|}<br />
----<br />
<br />
</div><br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/TeamTeam:Nevada/Team2010-10-25T04:19:49Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Picture Team.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
<p style="text-align:left;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Meet the Team</span><br />
</p><br />
----<br />
<div style="padding: 20px 0 25px 25px;"><br />
<br />
<html><br />
<a href="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG" class="shadow" style="float:left;margin:0px;width:600px"></a><br />
</html><br />
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<br />
<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Advisors</span><br />
</p><br />
----<br />
<br />
[[Image:Christie.jpg|left|thumb|alt=alt text|Dr. Christie Howard]]<br />
<br />
[[Image:Shintani.jpg|left|thumb|alt=alt text|Dr. Dave Shintani]]<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
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<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Undergraduates</span><br />
</p><br />
----<br />
<gallery widths=170px heights=170px perrow=4><br />
Image:Hilary UNR.png|Hilary Allen<br />
Image:mpolasko2.JPG|Matthew Polasko<br />
Image:Randy.jpg|Randy Pares<br />
Image:Copley.jpg|Christian Copley<br />
Image:Bryson UNR 2.png|Bryson Wheeler<br />
Image:Gladwill.jpg|Vadim Gladwill<br />
Image:Image-Elaine.jpeg|Elaine Bersaba<br />
Image:Sam UNR .png|Samantha Lee<br />
Image:Nick UNR.png|Nick Noel<br />
Image:Hileary.jpg|Richard Hilleary<br />
</gallery><br />
<br />
<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:normal;text-decoration:bold;color:#15317E;background-color:ffffff;">What We Did</span><br />
</p><br />
----<br />
<br />
<div style="padding:10px;clear:both"><br />
{| border="0" cellpadding="0" cellspacing="20"<br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Team President</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">NT Cell Transformations</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter + Plant Kozak + RFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + EYFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + mCherry</span><br />
*<span style="font-size:12pt;font-weight:bold;">Composite - 35s Promoter + Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;"> DREB1C Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">35S Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Modeling</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">RD29A Promoter</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|}<br />
----<br />
<br />
</div><br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Mpolaskohttp://2010.igem.org/Team:Nevada/TeamTeam:Nevada/Team2010-10-25T04:16:24Z<p>Mpolasko: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Picture Team.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
<p style="text-align:left;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Meet the Team</span><br />
</p><br />
----<br />
<div style="padding: 20px 0 25px 25px;"><br />
<br />
<html><br />
<a href="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG" class="shadow" style="float:left;margin:0px;width:600px"></a><br />
</html><br />
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<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Advisors</span><br />
</p><br />
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[[Image:Christie.jpg|left|thumb|alt=alt text|Dr. Christie Howard]]<br />
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[[Image:Shintani.jpg|left|thumb|alt=alt text|Dr. Dave Shintani]]<br />
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<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Undergraduates</span><br />
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<gallery widths=170px heights=170px perrow=4><br />
Image:Hilary UNR.png|Hilary Allen<br />
Image:mpolasko2.JPG|Matthew Polasko<br />
Image:Randy.jpg|Randy Pares<br />
Image:Copley.jpg|Christian Copley<br />
Image:Bryson UNR 2.png|Bryson Wheeler<br />
Image:Gladwill.jpg|Vadim Gladwill<br />
Image:Image-Elaine.jpeg|Elaine Bersaba<br />
Image:Sam UNR .png|Samantha Lee<br />
Image:Nick UNR.png|Nick Noel<br />
Image:Hileary.jpg|Richard Hilleary<br />
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<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:normal;text-decoration:bold;color:#15317E;background-color:ffffff;">What We Did</span><br />
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{| border="0" cellpadding="0" cellspacing="20"<br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#2B60DE;background-color:ffffff;">Hilary Allen</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#2B60DE;background-color:ffffff;">Elaine Bersaba</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#2B60DE;background-color:ffffff;">Matthew Polasko</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#2B60DE;background-color:ffffff;">Christian Copley</span><br />
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|<br />
*<span style="font-size:12pt;font-weight:bold;">Team President</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">NT Cell Transformations</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
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*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter + Plant Kozak + RFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
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*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + EYFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + mCherry</span><br />
*<span style="font-size:12pt;font-weight:bold;">Composite - 35s Promoter + Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;"> DREB1C Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
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|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#2B60DE;background-color:ffffff;">Bryson Wheeler</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#2B60DE;background-color:ffffff;">Richard Hilleary</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#2B60DE;background-color:ffffff;">Nick Noel</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#2B60DE;background-color:ffffff;">Samantha Lee</span><br />
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*<span style="font-size:12pt;font-weight:bold;">35S Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Modeling</span><br />
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*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">RD29A Promoter</span><br />
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|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#2B60DE;background-color:ffffff;">Randy Pares</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#2B60DE;background-color:ffffff;">Vadim Gladwill</span><br />
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*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
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*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|}<br />
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!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
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|}</div>Mpolasko