http://2010.igem.org/wiki/index.php?title=Special:Contributions/Hilarya&feed=atom&limit=50&target=Hilarya&year=&month=2010.igem.org - User contributions [en]2024-03-28T09:57:56ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Nevada/DREB1CTeam:Nevada/DREB1C2010-10-28T03:12:38Z<p>Hilarya: </p>
<hr />
<div>{{nevadaDREB1C}}<br />
<br />
== Promoters ==<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 />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png '''DREB1C promoter''' [[Team:Nevada/registry submissions]] <br />
----<br />
<br />
<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 />
<br />
<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 />
<br />
<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 />
<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/DREB1CTeam:Nevada/DREB1C2010-10-28T03:12:25Z<p>Hilarya: /* Promoters */</p>
<hr />
<div>{{nevadaDREB1C}}<br />
<br />
<br />
== Promoters ==<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 />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png '''DREB1C promoter''' [[Team:Nevada/registry submissions]] <br />
----<br />
<br />
<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 />
<br />
<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 />
<br />
<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 />
<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/ModelingTeam:Nevada/Modeling2010-10-28T02:38:21Z<p>Hilarya: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Modeling.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
== Modeling ==<br />
<br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png<br />
----<br />
'''Introduction'''<br />
<br />
<p>Plant stress responses are often cascades involving hundreds of genes and gene products. The possible interactions in these cascades are astronomical. Therefore, the 2010 Nevada iGEM team worked with Bioinformatics Professor, Karen Schlauch, to use a computational method that could quickly analyze possible transcriptional regulation pathways, using either microarray data or data from continuous fluorometry experiments. In conjunction with the use of tobacco BY-2 (NT1) cells, this method could allow for even greater time efficiency in identifying important aspects of gene networks in plants. The method was intended to allow for easier identification of promoters useful to the team’s objective of creating remote plant biosensors. The method uses a Boolean network approach to examine the gene network and its possible regulatory system. We first viewed transcripts as “on” when above a threshold value and “off” for lesser values. All Boolean networks that could generate our dataset were generated and evaluated. In this manner, we were able to look at all possible interactions between genes based on the Boolean approach.</p><br />
<br />
'''Data'''<br />
<br />
<p>When it became clear that the team would not have sufficient time to perform fluorometry experiments and analyze data by November, it was decided that microarray experiments published to internet databases would have to do. All data was originally obtained from a 24-hour time course microarray experiment performed by Jian-Kang Zhu, ''et al,'' and published on the Gene Expression Omnibus database (Zhu, ''et al.'' 2005). This allowed for a proof-of-concept to see if the Boolean network would support what was known about the DREB1 pathway from published literature. <br />
<br><br />
<br>[https://2010.igem.org/Team:Nevada/Original_Data<u>Click here to see the initial data set</u>]<br />
<br><br />
<br>Because this data consisted of only four time points, all eight genes had similar Boolean values at each time point. Therefore, the Boolean functions for each were essentially the same and numbered in the billions. This provided little data for interpretation. Several methods were used to tease out differences in expression, so that the time courses would be sufficiently different. First, the threshold value for "on" was raised to 2^3.5 rather than 4. Second, The data was interpolated to estimate extra time points within the 24-hour time course. Finally, the number of inputs for each gene was limited to four, as it was deemed unlikely that any gene in this network was receiving input from 5 or more.<br />
<br><br />
<br>[https://2010.igem.org/Team:Nevada/Interpolated_Data<u>Click here to see the interpolated data set and associated Boolean functions</u>]</p><br />
<br />
'''Results'''<br />
<br />
<p>The data from the Boolean functions was used with knowledge from literature concerning cold-stress in ''Arabidopsis''. The literature showed that DREB1A, DREB1B and DREB1C were all known to have a role in cold-stress, that they acted on RD29A, and that they possibly acted on RAP2.1, RAP2.6 and ZAT6 (Yamaguchi-Shinozaki, K. 2006). Literature also showed that ICE1 seemed to have a significant role in regulating DREB1A, but may not interact strongly with DREB1B or DREB1C (Zhu J-K. 2005). From this information, a rough idea of what the network might look like was made. <br />
<br> <br />
<br>As mentioned elsewhere, the Boolean functions for the initial data set were too numerous to work with. Curves were fit to the few time points available from the initial data set. The Boolean functions from this new data set did provide some useful information. It does support the hypothesis that all three of the DREB1 genes in the network are feeding into RD29A, with the Boolean function: 4 ( 2 ) & ( 3 ) & ( 4 ) & ( 6 ), where 2, 3, and 4 are DREB1s A, B and C respectively and 6 is ZAT6. The functions also showed that DREB1A was not receiving input from any gene in this network, indicating that DREB1A is probably not regulated by DREB1B or DREB1C. It was assumed that no input into DREB1A from ICE1 was observed because of difficulty in determining when ICE1 was active or inactive. The Boolean functions, as well as the expression data, indicate that RAP2.6 is not connected to this network. However, it is possible that RAP2.6 is primarily regulated post-transcriptionally, which would not be picked up by the Boolean network. Or, it may be an extremely late-response element only becoming active after 24 hours of freezing, and its increased expression at times after 24 hours may not have been adequately described by extrapolation. <br />
<br><br />
<br>It must be noted that the data used in the Boolean networking was not ideal. More time points would have greatly increased the efficacy, stressing the desirability of continuous fluorometry experiments. With the interpolated data, there may have been gross inaccuracies produced, which may have caused functions of the real network to be eliminated. On the upside, this provides much room for refining the method, should any future teams attempt to use a Boolean approach in computations. The Nevada team’s advice would be: use as many genes from a network as are feasible, run microarray/fluorometry experiments at least in triplicate, collect data at many time points or preferably continuously, and before running the computations, minimize the number of possible connections by ruling out relationships between genes that are known to be false. <br />
<br><br />
<br><html><a href="https://static.igem.org/mediawiki/2010/7/7e/IGEM_DREB1_pathway2.PNG"><img src="https://static.igem.org/mediawiki/2010/7/7e/IGEM_DREB1_pathway2.PNG" style="float:center;width:400px;margin:10px"></a></html></p><br />
<br><br />
'''Acknowledgments'''<br />
<br />
<p>The 2010 Nevada iGEM team would like to thank '''Karen Schlauch''' for all of her hard work, performing computational analysis, explaining the concepts of Boolean networking, and working with the team to find biological meaning in the Boolean output functions.</p><br />
<br />
'''References'''<br />
<br />
<p>'''Yamaguchi-Shinozaki, K., Nakashima, K.''' (2006) Regulons involved in osmotic stress-responsive and stress-responsive gene expression in plants. ''Physiologia Plantarum,'' 126: 62-71.<br />
<br><br />
'''Zhu, J-K., Lee, B., Henderson, D.''' (2005) The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1. ''Plant Cell.'' Vol. 17, Issue 11, p3155-3175.</p><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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/ModelingTeam:Nevada/Modeling2010-10-28T02:37:44Z<p>Hilarya: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Modeling.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
== Modeling ==<br />
<br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png<br />
----<br />
'''Introduction'''<br />
<br />
<p>Plant stress responses are often cascades involving hundreds of genes and gene products. The possible interactions in these cascades are astronomical. Therefore, the 2010 Nevada iGEM team worked with Bioinformatics Professor, Karen Schlauch, to use a computational method that could quickly analyze possible transcriptional regulation pathways, using either microarray data or data from continuous fluorometry experiments. In conjunction with the use of tobacco BY-2 (NT1) cells, this method could allow for even greater time efficiency in identifying important aspects of gene networks in plants. The method was intended to allow for easier identification of promoters useful to the team’s objective of creating remote plant biosensors. The method uses a Boolean network approach to examine the gene network and its possible regulatory system. We first viewed transcripts as “on” when above a threshold value and “off” for lesser values. All Boolean networks that could generate our dataset were generated and evaluated. In this manner, we were able to look at all possible interactions between genes based on the Boolean approach.</p><br />
<br />
'''Data'''<br />
<br />
<p>When it became clear that the team would not have sufficient time to perform fluorometry experiments and analyze data by November, it was decided that microarray experiments published to internet databases would have to do. All data was originally obtained from a 24-hour time course microarray experiment performed by Jian-Kang Zhu, ''et al,'' and published on the Gene Expression Omnibus database (Zhu, ''et al.'' 2005). This allowed for a proof-of-concept to see if the Boolean network would support what was known about the DREB1 pathway from published literature. <br />
<br><br />
<br>[https://2010.igem.org/Team:Nevada/Original_Data<u>Click here to see the initial data set</u>]<br />
<br><br />
<br>Because this data consisted of only four time points, all eight genes had similar Boolean values at each time point. Therefore, the Boolean functions for each were essentially the same and numbered in the billions. This provided little data for interpretation. Several methods were used to tease out differences in expression, so that the time courses would be sufficiently different. First, the threshold value for "on" was raised to 2^3.5 rather than 4. Second, The data was interpolated to estimate extra time points within the 24-hour time course. Finally, the number of inputs for each gene was limited to four, as it was deemed unlikely that any gene in this network was receiving input from 5 or more.<br />
<br><br />
<br>[https://2010.igem.org/Team:Nevada/Interpolated_Data<u>Click here to see the interpolated data set and associated Boolean functions</u>]</p><br />
<br><br />
<br><br />
'''Results'''<br />
<br />
<p>The data from the Boolean functions was used with knowledge from literature concerning cold-stress in ''Arabidopsis''. The literature showed that DREB1A, DREB1B and DREB1C were all known to have a role in cold-stress, that they acted on RD29A, and that they possibly acted on RAP2.1, RAP2.6 and ZAT6 (Yamaguchi-Shinozaki, K. 2006). Literature also showed that ICE1 seemed to have a significant role in regulating DREB1A, but may not interact strongly with DREB1B or DREB1C (Zhu J-K. 2005). From this information, a rough idea of what the network might look like was made. <br />
<br> <br />
<br>As mentioned elsewhere, the Boolean functions for the initial data set were too numerous to work with. Curves were fit to the few time points available from the initial data set. The Boolean functions from this new data set did provide some useful information. It does support the hypothesis that all three of the DREB1 genes in the network are feeding into RD29A, with the Boolean function: 4 ( 2 ) & ( 3 ) & ( 4 ) & ( 6 ), where 2, 3, and 4 are DREB1s A, B and C respectively and 6 is ZAT6. The functions also showed that DREB1A was not receiving input from any gene in this network, indicating that DREB1A is probably not regulated by DREB1B or DREB1C. It was assumed that no input into DREB1A from ICE1 was observed because of difficulty in determining when ICE1 was active or inactive. The Boolean functions, as well as the expression data, indicate that RAP2.6 is not connected to this network. However, it is possible that RAP2.6 is primarily regulated post-transcriptionally, which would not be picked up by the Boolean network. Or, it may be an extremely late-response element only becoming active after 24 hours of freezing, and its increased expression at times after 24 hours may not have been adequately described by extrapolation. <br />
<br><br />
<br>It must be noted that the data used in the Boolean networking was not ideal. More time points would have greatly increased the efficacy, stressing the desirability of continuous fluorometry experiments. With the interpolated data, there may have been gross inaccuracies produced, which may have caused functions of the real network to be eliminated. On the upside, this provides much room for refining the method, should any future teams attempt to use a Boolean approach in computations. The Nevada team’s advice would be: use as many genes from a network as are feasible, run microarray/fluorometry experiments at least in triplicate, collect data at many time points or preferably continuously, and before running the computations, minimize the number of possible connections by ruling out relationships between genes that are known to be false. <br />
<br><br />
<br><html><a href="https://static.igem.org/mediawiki/2010/7/7e/IGEM_DREB1_pathway2.PNG"><img src="https://static.igem.org/mediawiki/2010/7/7e/IGEM_DREB1_pathway2.PNG" style="float:center;width:400px;margin:10px"></a></html></p><br />
<br><br />
'''Acknowledgments'''<br />
<br />
<p>The 2010 Nevada iGEM team would like to thank '''Karen Schlauch''' for all of her hard work, performing computational analysis, explaining the concepts of Boolean networking, and working with the team to find biological meaning in the Boolean output functions.</p><br />
<br><br />
<br><br />
'''References'''<br />
<br />
<p>'''Yamaguchi-Shinozaki, K., Nakashima, K.''' (2006) Regulons involved in osmotic stress-responsive and stress-responsive gene expression in plants. ''Physiologia Plantarum,'' 126: 62-71.<br />
<br><br />
'''Zhu, J-K., Lee, B., Henderson, D.''' (2005) The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1. ''Plant Cell.'' Vol. 17, Issue 11, p3155-3175.</p><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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/ModelingTeam:Nevada/Modeling2010-10-28T02:35:10Z<p>Hilarya: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Modeling.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
== Modeling ==<br />
<br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png<br />
----<br />
'''Introduction'''<br />
<br />
<p>Plant stress responses are often cascades involving hundreds of genes and gene products. The possible interactions in these cascades are astronomical. Therefore, the 2010 Nevada iGEM team worked with Bioinformatics Professor, Karen Schlauch, to use a computational method that could quickly analyze possible transcriptional regulation pathways, using either microarray data or data from continuous fluorometry experiments. In conjunction with the use of tobacco BY-2 (NT1) cells, this method could allow for even greater time efficiency in identifying important aspects of gene networks in plants. The method was intended to allow for easier identification of promoters useful to the team’s objective of creating remote plant biosensors. The method uses a Boolean network approach to examine the gene network and its possible regulatory system. We first viewed transcripts as “on” when above a threshold value and “off” for lesser values. All Boolean networks that could generate our dataset were generated and evaluated. In this manner, we were able to look at all possible interactions between genes based on the Boolean approach.</p><br />
<br />
'''Data'''<br />
<br><br />
When it became clear that the team would not have sufficient time to perform fluorometry experiments and analyze data by November, it was decided that microarray experiments published to internet databases would have to do. All data was originally obtained from a 24-hour time course microarray experiment performed by Jian-Kang Zhu, ''et al,'' and published on the Gene Expression Omnibus database (Zhu, ''et al.'' 2005). This allowed for a proof-of-concept to see if the Boolean network would support what was known about the DREB1 pathway from published literature. <br />
<br><br />
<br>[https://2010.igem.org/Team:Nevada/Original_Data<u>Click here to see the initial data set</u>]<br />
<br><br />
<br>Because this data consisted of only four time points, all eight genes had similar Boolean values at each time point. Therefore, the Boolean functions for each were essentially the same and numbered in the billions. This provided little data for interpretation. Several methods were used to tease out differences in expression, so that the time courses would be sufficiently different. First, the threshold value for "on" was raised to 2^3.5 rather than 4. Second, The data was interpolated to estimate extra time points within the 24-hour time course. Finally, the number of inputs for each gene was limited to four, as it was deemed unlikely that any gene in this network was receiving input from 5 or more.<br />
<br><br />
<br>[https://2010.igem.org/Team:Nevada/Interpolated_Data<u>Click here to see the interpolated data set and associated Boolean functions</u>]<br />
<br><br />
<br><br />
'''Results'''<br />
<br><br />
<br>The data from the Boolean functions was used with knowledge from literature concerning cold-stress in ''Arabidopsis''. The literature showed that DREB1A, DREB1B and DREB1C were all known to have a role in cold-stress, that they acted on RD29A, and that they possibly acted on RAP2.1, RAP2.6 and ZAT6 (Yamaguchi-Shinozaki, K. 2006). Literature also showed that ICE1 seemed to have a significant role in regulating DREB1A, but may not interact strongly with DREB1B or DREB1C (Zhu J-K. 2005). From this information, a rough idea of what the network might look like was made. <br />
<br> <br />
<br>As mentioned elsewhere, the Boolean functions for the initial data set were too numerous to work with. Curves were fit to the few time points available from the initial data set. The Boolean functions from this new data set did provide some useful information. It does support the hypothesis that all three of the DREB1 genes in the network are feeding into RD29A, with the Boolean function: 4 ( 2 ) & ( 3 ) & ( 4 ) & ( 6 ), where 2, 3, and 4 are DREB1s A, B and C respectively and 6 is ZAT6. The functions also showed that DREB1A was not receiving input from any gene in this network, indicating that DREB1A is probably not regulated by DREB1B or DREB1C. It was assumed that no input into DREB1A from ICE1 was observed because of difficulty in determining when ICE1 was active or inactive. The Boolean functions, as well as the expression data, indicate that RAP2.6 is not connected to this network. However, it is possible that RAP2.6 is primarily regulated post-transcriptionally, which would not be picked up by the Boolean network. Or, it may be an extremely late-response element only becoming active after 24 hours of freezing, and its increased expression at times after 24 hours may not have been adequately described by extrapolation. <br />
<br><br />
<br>It must be noted that the data used in the Boolean networking was not ideal. More time points would have greatly increased the efficacy, stressing the desirability of continuous fluorometry experiments. With the interpolated data, there may have been gross inaccuracies produced, which may have caused functions of the real network to be eliminated. On the upside, this provides much room for refining the method, should any future teams attempt to use a Boolean approach in computations. The Nevada team’s advice would be: use as many genes from a network as are feasible, run microarray/fluorometry experiments at least in triplicate, collect data at many time points or preferably continuously, and before running the computations, minimize the number of possible connections by ruling out relationships between genes that are known to be false. <br />
<br><br />
<br><html><a href="https://static.igem.org/mediawiki/2010/7/7e/IGEM_DREB1_pathway2.PNG"><img src="https://static.igem.org/mediawiki/2010/7/7e/IGEM_DREB1_pathway2.PNG" style="float:center;width:400px;margin:10px"></a></html><br />
<br><br />
'''Acknowledgments'''<br />
<br><br />
<br>The 2010 Nevada iGEM team would like to thank '''Karen Schlauch''' for all of her hard work, performing computational analysis, explaining the concepts of Boolean networking, and working with the team to find biological meaning in the Boolean output functions.<br />
<br><br />
<br><br />
'''References'''<br />
<br><br />
<br><br />
'''Yamaguchi-Shinozaki, K., Nakashima, K.''' (2006) Regulons involved in osmotic stress-responsive and stress-responsive gene expression in plants. ''Physiologia Plantarum,'' 126: 62-71.<br />
<br><br />
'''Zhu, J-K., Lee, B., Henderson, D.''' (2005) The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1. ''Plant Cell.'' Vol. 17, Issue 11, p3155-3175.<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/ModelingTeam:Nevada/Modeling2010-10-28T02:32:20Z<p>Hilarya: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Modeling.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
== Modeling ==<br />
<br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png<br />
----<br />
'''Introduction'''<br />
<br />
<p>Plant stress responses are often cascades involving hundreds of genes and gene products. The possible interactions in these cascades are astronomical. Therefore, the 2010 Nevada iGEM team worked with Bioinformatics Professor, Karen Schlauch, to use a computational method that could quickly analyze possible transcriptional regulation pathways, using either microarray data or data from continuous fluorometry experiments. In conjunction with the use of tobacco BY-2 (NT1) cells, this method could allow for even greater time efficiency in identifying important aspects of gene networks in plants. The method was intended to allow for easier identification of promoters useful to the team’s objective of creating remote plant biosensors. The method uses a Boolean network approach to examine the gene network and its possible regulatory system. We first viewed transcripts as “on” when above a threshold value and “off” for lesser values. All Boolean networks that could generate our dataset were generated and evaluated. In this manner, we were able to look at all possible interactions between genes based on the Boolean approach.</p><br />
<br />
'''Data'''<br />
<br><br />
When it became clear that the team would not have sufficient time to perform fluorometry experiments and analyze data by November, it was decided that microarray experiments published to internet databases would have to do. All data was originally obtained from a 24-hour time course microarray experiment performed by Jian-Kang Zhu, ''et al,'' and published on the Gene Expression Omnibus database (Zhu, ''et al.'' 2005). This allowed for a proof-of-concept to see if the Boolean network would support what was known about the DREB1 pathway from published literature. <br />
<br><br />
<br>[https://2010.igem.org/Team:Nevada/Original_Data<u>Click here to see the initial data set</u>]<br />
<br><br />
<br>Because this data consisted of only four time points, all eight genes had similar Boolean values at each time point. Therefore, the Boolean functions for each were essentially the same and numbered in the billions. This provided little data for interpretation. Several methods were used to tease out differences in expression, so that the time courses would be sufficiently different. First, the threshold value for "on" was raised to 2^3.5 rather than 4. Second, The data was interpolated to estimate extra time points within the 24-hour time course. Finally, the number of inputs for each gene was limited to four, as it was deemed unlikely that any gene in this network was receiving input from 5 or more.<br />
<br><br />
<br>[https://2010.igem.org/Team:Nevada/Interpolated_Data<u>Click here to see the interpolated data set and associated Boolean functions</u>]<br />
<br><br />
'''Results'''<br />
<br><br />
<br>The data from the Boolean functions was used with knowledge from literature concerning cold-stress in ''Arabidopsis''. The literature showed that DREB1A, DREB1B and DREB1C were all known to have a role in cold-stress, that they acted on RD29A, and that they possibly acted on RAP2.1, RAP2.6 and ZAT6 (Yamaguchi-Shinozaki, K. 2006). Literature also showed that ICE1 seemed to have a significant role in regulating DREB1A, but may not interact strongly with DREB1B or DREB1C (Zhu J-K. 2005). From this information, a rough idea of what the network might look like was made. <br />
<br> <br />
<br>As mentioned elsewhere, the Boolean functions for the initial data set were too numerous to work with. Curves were fit to the few time points available from the initial data set. The Boolean functions from this new data set did provide some useful information. It does support the hypothesis that all three of the DREB1 genes in the network are feeding into RD29A, with the Boolean function: 4 ( 2 ) & ( 3 ) & ( 4 ) & ( 6 ), where 2, 3, and 4 are DREB1s A, B and C respectively and 6 is ZAT6. The functions also showed that DREB1A was not receiving input from any gene in this network, indicating that DREB1A is probably not regulated by DREB1B or DREB1C. It was assumed that no input into DREB1A from ICE1 was observed because of difficulty in determining when ICE1 was active or inactive. The Boolean functions, as well as the expression data, indicate that RAP2.6 is not connected to this network. However, it is possible that RAP2.6 is primarily regulated post-transcriptionally, which would not be picked up by the Boolean network. Or, it may be an extremely late-response element only becoming active after 24 hours of freezing, and its increased expression at times after 24 hours may not have been adequately described by extrapolation. <br />
<br><br />
<br>It must be noted that the data used in the Boolean networking was not ideal. More time points would have greatly increased the efficacy, stressing the desirability of continuous fluorometry experiments. With the interpolated data, there may have been gross inaccuracies produced, which may have caused functions of the real network to be eliminated. On the upside, this provides much room for refining the method, should any future teams attempt to use a Boolean approach in computations. The Nevada team’s advice would be: use as many genes from a network as are feasible, run microarray/fluorometry experiments at least in triplicate, collect data at many time points or preferably continuously, and before running the computations, minimize the number of possible connections by ruling out relationships between genes that are known to be false. <br />
<br><br />
<br><html><a href="https://static.igem.org/mediawiki/2010/7/7e/IGEM_DREB1_pathway2.PNG"><img src="https://static.igem.org/mediawiki/2010/7/7e/IGEM_DREB1_pathway2.PNG" style="float:center;width:400px;margin:10px"></a></html><br />
<br><br />
'''Acknowledgments'''<br />
<br><br />
<br>The 2010 Nevada iGEM team would like to thank '''Karen Schlauch''' for all of her hard work, performing computational analysis, explaining the concepts of Boolean networking, and working with the team to find biological meaning in the Boolean output functions.<br />
<br><br />
<br><br />
'''References'''<br />
<br><br />
'''Yamaguchi-Shinozaki, K., Nakashima, K.''' (2006) Regulons involved in osmotic stress-responsive and stress-responsive gene expression in plants. ''Physiologia Plantarum,'' 126: 62-71.<br />
<br><br />
'''Zhu, J-K., Lee, B., Henderson, D.''' (2005) The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1. ''Plant Cell.'' Vol. 17, Issue 11, p3155-3175.<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/ModelingTeam:Nevada/Modeling2010-10-28T02:22:38Z<p>Hilarya: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Modeling.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
'''Modeling''' <br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png<br />
----<br />
'''Introduction'''<br />
<br />
<p>Plant stress responses are often cascades involving hundreds of genes and gene products. The possible interactions in these cascades are astronomical. Therefore, the 2010 Nevada iGEM team worked with Bioinformatics Professor, Karen Schlauch, to use a computational method that could quickly analyze possible transcriptional regulation pathways, using either microarray data or data from continuous fluorometry experiments. In conjunction with the use of tobacco BY-2 (NT1) cells, this method could allow for even greater time efficiency in identifying important aspects of gene networks in plants. The method was intended to allow for easier identification of promoters useful to the team’s objective of creating remote plant biosensors. The method uses a Boolean network approach to examine the gene network and its possible regulatory system. We first viewed transcripts as “on” when above a threshold value and “off” for lesser values. All Boolean networks that could generate our dataset were generated and evaluated. In this manner, we were able to look at all possible interactions between genes based on the Boolean approach.</p><br />
<br />
=== Boolean Networks ===<br />
<br />
'''Data'''<br />
<br><br />
When it became clear that the team would not have sufficient time to perform fluorometry experiments and analyze data by November, it was decided that microarray experiments published to internet databases would have to do. All data was originally obtained from a 24-hour time course microarray experiment performed by Jian-Kang Zhu, ''et al,'' and published on the Gene Expression Omnibus database (Zhu, ''et al.'' 2005). This allowed for a proof-of-concept to see if the Boolean network would support what was known about the DREB1 pathway from published literature. <br />
<br><br />
<br>[https://2010.igem.org/Team:Nevada/Original_Data<u>Click here to see the initial data set</u>]<br />
<br><br />
<br>Because this data consisted of only four time points, all eight genes had similar Boolean values at each time point. Therefore, the Boolean functions for each were essentially the same and numbered in the billions. This provided little data for interpretation. Several methods were used to tease out differences in expression, so that the time courses would be sufficiently different. First, the threshold value for "on" was raised to 2^3.5 rather than 4. Second, The data was interpolated to estimate extra time points within the 24-hour time course. Finally, the number of inputs for each gene was limited to four, as it was deemed unlikely that any gene in this network was receiving input from 5 or more.<br />
<br><br />
<br>[https://2010.igem.org/Team:Nevada/Interpolated_Data<u>Click here to see the interpolated data set and associated Boolean functions</u>]<br />
<br><br />
'''Results'''<br />
<br><br />
<br>The data from the Boolean functions was used with knowledge from literature concerning cold-stress in ''Arabidopsis''. The literature showed that DREB1A, DREB1B and DREB1C were all known to have a role in cold-stress, that they acted on RD29A, and that they possibly acted on RAP2.1, RAP2.6 and ZAT6 (Yamaguchi-Shinozaki, K. 2006). Literature also showed that ICE1 seemed to have a significant role in regulating DREB1A, but may not interact strongly with DREB1B or DREB1C (Zhu J-K. 2005). From this information, a rough idea of what the network might look like was made. <br />
<br> <br />
<br>As mentioned elsewhere, the Boolean functions for the initial data set were too numerous to work with. Curves were fit to the few time points available from the initial data set. The Boolean functions from this new data set did provide some useful information. It does support the hypothesis that all three of the DREB1 genes in the network are feeding into RD29A, with the Boolean function: 4 ( 2 ) & ( 3 ) & ( 4 ) & ( 6 ), where 2, 3, and 4 are DREB1s A, B and C respectively and 6 is ZAT6. The functions also showed that DREB1A was not receiving input from any gene in this network, indicating that DREB1A is probably not regulated by DREB1B or DREB1C. It was assumed that no input into DREB1A from ICE1 was observed because of difficulty in determining when ICE1 was active or inactive. The Boolean functions, as well as the expression data, indicate that RAP2.6 is not connected to this network. However, it is possible that RAP2.6 is primarily regulated post-transcriptionally, which would not be picked up by the Boolean network. Or, it may be an extremely late-response element only becoming active after 24 hours of freezing, and its increased expression at times after 24 hours may not have been adequately described by extrapolation. <br />
<br><br />
<br>It must be noted that the data used in the Boolean networking was not ideal. More time points would have greatly increased the efficacy, stressing the desirability of continuous fluorometry experiments. With the interpolated data, there may have been gross inaccuracies produced, which may have caused functions of the real network to be eliminated. On the upside, this provides much room for refining the method, should any future teams attempt to use a Boolean approach in computations. The Nevada team’s advice would be: use as many genes from a network as are feasible, run microarray/fluorometry experiments at least in triplicate, collect data at many time points or preferably continuously, and before running the computations, minimize the number of possible connections by ruling out relationships between genes that are known to be false. <br />
<br><br />
<br><html><a href="https://static.igem.org/mediawiki/2010/7/7e/IGEM_DREB1_pathway2.PNG"><img src="https://static.igem.org/mediawiki/2010/7/7e/IGEM_DREB1_pathway2.PNG" style="float:center;width:400px;margin:10px"></a></html><br />
<br><br />
'''Acknowledgments'''<br />
<br><br />
<br>The 2010 Nevada iGEM team would like to thank '''Karen Schlauch''' for all of her hard work, performing computational analysis, explaining the concepts of Boolean networking, and working with the team to find biological meaning in the Boolean output functions.<br />
<br><br />
'''References'''<br />
<br><br />
'''Yamaguchi-Shinozaki, K., Nakashima, K.''' (2006) Regulons involved in osmotic stress-responsive and stress-responsive gene expression in plants. ''Physiologia Plantarum,'' 126: 62-71.<br />
<br><br />
'''Zhu, J-K., Lee, B., Henderson, D.''' (2005) The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1. ''Plant Cell.'' Vol. 17, Issue 11, p3155-3175.<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/Transgenic_PlantsTeam:Nevada/Transgenic Plants2010-10-28T00:21:23Z<p>Hilarya: /* Transgenic Plants: into the Wild */</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 />
<p>'''Technological Advances from Genetically Engineered Plants'''<br />
<br><br />
Since the initial development of Agrobacterium transformation systems, many plant species including tobacco, tomato, potato, rice, soybean, mint, melon, cucumber, pine and poplar trees, and many others have been transformed using this ingenious bacterial vector. Important traits have been engineered into plants including pest and weed resistance, increased nutritional value, environmental stress tolerance, the production of pharmaceutical and industrial proteins, and the production of bioactive secondary chemical compounds. Our ability to genetically engineer plants has revolutionized agriculture by increasing crop yields while drastically decreasing the application of herbicides and pesticides. This technology is necessary to allow farmers to produce sufficient food for a growing global population. Furthermore, plants are currently being engineered to produce fuel and chemical alternatives to petroleum based products. Because plants are net consumers of atmospheric carbon dioxide, they are currently being seen as a means to sequester greenhouse gases while at the same time replacing petroleum and coal as chemical feedstocks. <br />
<br><br />
<br>'''Concerns Related to the Use of GMO Plants:'''<br />
Although transgenic plants have made a large positive impact on modern agriculture, the use of GMO crops is still a very controversial topic. Issues related to GMOS and health, the environment, economics and ethics are of concern to many people. <br />
<br><br />
*'''Health Concerns''' – While the introduction of genetically engineered traits has made a huge impact on crop yields, many people are concerned about the introduction of transgenes into the human food chain. Although extensive testing of GMO crops is required by the FDA and USDA before approval for cultivation, some people still worry that GMO foods are “unnatural” and may cause health problems when consumed. Many argue that even though health issues have not yet arisen, problems may occur in the future. <br />
<br><br />
*'''Environmental Concerns''' – The widespread use of GMO crops has concerned environmentalist on a number of levels. First of all, many see the use of herbicide resistant crops as being harmful to the environment because farmers will be prone to increase their use of specific herbicide to control weeds. People also worry about the possible transfer of the herbicide resistant genes to closely related weedy plant species could lead to the development of superweeds. Concerns have also arisen from the expression of insecticidal genes that may lead to the killing of not only pest insects, but also beneficial insects such as pollinators or predatory insects. <br />
<br><br />
*'''Economic Concerns''' – Many object to the fact that the distribution of GMO seed is controlled by large multinational conglomerates. And that due to proprietary issues, ownership of the seed, even in later generations, remains the property of these companies. Therefore farmers can no longer save seed from one year’s harvest to plant as next year’s crop. While this has been an issue since the advent of hybrid seeds in the 1950s and 1960s, many object to these practices because they prevent small farmers from realizing the benefits of GMOs while at the same time practicing subsistence farming. <br />
<br><br />
*'''Ethical Concerns''' – In regards to people’s personal beliefs, some feel that manipulating plant genomes through recombinant DNA approaches is an abomination to their god. They see this as an unnatural act that goes against the higher order of things. <br />
<br />
<br>While many of these concerns have been addressed through technological developments and stringent government regulations, many people will always reject GMOs due to conflicts with basic belief systems. While it is difficult to convince such people that the benefits of GMO plants outweigh the concerns, our job as scientist is to alleviate their concerns with rational science based dialog. <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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/CompositeTeam:Nevada/Composite2010-10-28T00:17:22Z<p>Hilarya: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Composite 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 />
== 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 />
<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/File:Composite_UNR_Final.pngFile:Composite UNR Final.png2010-10-28T00:16:38Z<p>Hilarya: uploaded a new version of "Image:Composite UNR Final.png"</p>
<hr />
<div></div>Hilaryahttp://2010.igem.org/File:Composite_UNR_Final.pngFile:Composite UNR Final.png2010-10-28T00:16:11Z<p>Hilarya: </p>
<hr />
<div></div>Hilaryahttp://2010.igem.org/Team:Nevada/Transgenic_PlantsTeam:Nevada/Transgenic Plants2010-10-28T00:12:31Z<p>Hilarya: /* Transgenic Plants: into the Wild */</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 />
<p>'''Technological Advances from Genetically Engineered Plants'''<br />
<br><br />
Since the initial development of Agrobacterium transformation systems, many plant species including tobacco, tomato, potato, rice, soybean, mint, melon, cucumber, pine and poplar trees, and many others have been transformed using this ingenious bacterial vector. Important traits have been engineered into plants including pest and weed resistance, increased nutritional value, environmental stress tolerance, the production of pharmaceutical and industrial proteins, and the production of bioactive secondary chemical compounds. Our ability to genetically engineer plants has revolutionized agriculture by increasing crop yields while drastically decreasing the application of herbicides and pesticides. This technology is necessary to allow farmers to produce sufficient food for a growing global population. Furthermore, plants are currently being engineered to produce fuel and chemical alternatives to petroleum based products. Because plants are net consumers of atmospheric carbon dioxide, they are currently being seen as a means to sequester greenhouse gases while at the same time replacing petroleum and coal as chemical feedstocks. <br />
<br><br />
<br>'''Concerns Related to the Use of GMO Plants:'''<br />
Although transgenic plants have made a large positive impact on modern agriculture, the use of GMO crops is still a very controversial topic. Issues related to GMOS and health, the environment, economics and ethics are of concern to many people. <br />
<br><br />
*'''Health Concerns''' – While the introduction of genetically engineered traits has made a huge impact on crop yields, many people are concerns about the introduction of transgenes into the human food chain. Although extensive testing of GMO crops is required by the FDA and USDA before approval for cultivation, some people still worry that GMO foods are “unnatural” and may cause health problems when consumed. Many argue that even though health issues have not yet arisen, problems may occur in the future. <br />
<br><br />
*'''Environmental Concerns''' – The widespread use of GMO crops has concerned environmentalist on a number of levels. First of all, many see the use of herbicide resistant crops as being harmful to the environment because farmers will be prone to increase their use of specific herbicide to control weeds. People also worry about the possible transfer of the herbicide resistant genes to closely related weedy plant species could lead to the development of superweeds. Concerns have also arisen from the expression of insecticidal genes that may lead to the killing of not only pest insects, but also beneficial insects such as pollinators or predatory insects. <br />
<br><br />
*'''Economic Concerns''' – Many object to the fact that the distribution of GMO seed is controlled by large multinational conglomerates. And that due to proprietary issues, ownership of the seed, even in later generations, remains the property of these companies. Therefore farmers can no longer save seed from one year’s harvest to plant as next year’s crop. While this has been an issue since the advent of hybrid seeds in the 1950s and 1960s, mainly object to these practices because they prevent small farmers from realizing the benefits of GMOs while at the same time practicing subsistence farming. <br />
<br><br />
*'''Ethical Concerns''' – In regards to people’s personal beliefs, some feel that manipulating plant genomes through recombinant DNA approaches is an abomination to their god. They see this as an unnatural act that goes against the higher order of things. <br />
<br />
<br>While many of these concerns have been address through technological developments and stringent government regulations, many people will always reject GMO due to conflicts with basic belief systems. While it is difficult to convince such people that the benefits of GMO plants outweigh the concerns, our job as scientist is to alleviate their concerns with rational science based dialog. <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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/Transgenic_PlantsTeam:Nevada/Transgenic Plants2010-10-28T00:12:09Z<p>Hilarya: /* Transgenic Plants: into the Wild */</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 />
<p>'''Technological Advances from Genetically Engineered Plants'''<br />
<br><br />
Since the initial development of Agrobacterium transformation systems, many plant species including tobacco, tomato, potato, rice, soybean, mint, melon, cucumber, pine and poplar trees, and many others have been transformed using this ingenious bacterial vector. Important traits have been engineered into plants including pest and weed resistance, increased nutritional value, environmental stress tolerance, the production of pharmaceutical and industrial proteins, and the production of bioactive secondary chemical compounds. Our ability to genetically engineer plants has revolutionized agriculture by increasing crop yields while drastically decreasing the application of herbicides and pesticides. This technology is necessary to allow farmers to produce sufficient food for a growing global population. Furthermore, plants are currently being engineered to produce fuel and chemical alternatives to petroleum based products. Because plants are net consumers of atmospheric carbon dioxide, they are currently being seen as a means to sequester greenhouse gases while at the same time replacing petroleum and coal as chemical feedstocks. <br />
<br><br />
<br>'''Concerns Related to the Use of GMO Plants:'''<br />
Although transgenic plants have made a large positive impact on modern agriculture, the use of GMO crops is still a very controversial topic. Issues related to GMOS and health, the environment, economics and ethics are of concern to many people. <br />
<br><br />
*'''Health Concerns''' – While the introduction of genetically engineered traits has made a huge impact on crop yields, many people are concerns about the introduction of transgenes into the human food chain. Although extensive testing of GMO crops is required by the FDA and USDA before approval for cultivation, some people still worry that GMO foods are “unnatural” and may cause health problems when consumed. Many argue that even though health issues have not yet arisen, problems may occur in the future. <br />
<br><br />
*'''Environmental Concerns''' – The widespread use of GMO crops has concerned environmentalist on a number of levels. First of all, many see the use of herbicide resistant crops as being harmful to the environment because farmers will be prone to increase their use of specific herbicide to control weeds. People also worry about the possible transfer of the herbicide resistant genes to closely related weedy plant species could lead to the development of superweeds. Concerns have also arisen from the expression of insecticidal genes that may lead to the killing of not only pest insects, but also beneficial insects such as pollinators or predatory insects. <br />
<br><br />
*'''Economic Concerns''' – Many object to the fact that the distribution of GMO seed is controlled by large multinational conglomerates. And that due to proprietary issues, ownership of the seed, even in later generations, remains the property of these companies. Therefore farmers can no longer save seed from one year’s harvest to plant as next year’s crop. While this has been an issue since the advent of hybrid seeds in the 1950s and 1960s, mainly object to these practices because they prevent small farmers from realizing the benefits of GMOs while at the same time practicing subsistence farming. <br />
<br><br />
*'''Ethical Concerns''' – In regards to people’s personal beliefs, some feel that manipulating plant genomes through recombinant DNA approaches is an abomination to their god. They see this as an unnatural act that goes against the higher order of things. <br />
<br />
<br>While many of these concerns have been address through technological developments and stringent government regulations, many people will always reject GMO due to conflicts with basic belief systems. While it is difficult to convince such people that the benefits of GMO plants outweigh the concerns, our job as scientist is to alleviate their concerns with rational science based dialog. <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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/Transgenic_PlantsTeam:Nevada/Transgenic Plants2010-10-28T00:11:18Z<p>Hilarya: /* Transgenic Plants: into the Wild */</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 />
<p>'''Technological Advances from Genetically Engineered Plants'''<br />
<br><br />
Since the initial development of Agrobacterium transformation systems, many plant species including tobacco, tomato, potato, rice, soybean, mint, melon, cucumber, pine and poplar trees, and many others have been transformed using this ingenious bacterial vector. Important traits have been engineered into plants including pest and weed resistance, increased nutritional value, environmental stress tolerance, the production of pharmaceutical and industrial proteins, and the production of bioactive secondary chemical compounds. Our ability to genetically engineer plants has revolutionized agriculture by increasing crop yields while drastically decreasing the application of herbicides and pesticides. This technology is necessary to allow farmers to produce sufficient food for a growing global population. Furthermore, plants are currently being engineered to produce fuel and chemical alternatives to petroleum based products. Because plants are net consumers of atmospheric carbon dioxide, they are currently being seen as a means to sequester greenhouse gases while at the same time replacing petroleum and coal as chemical feedstocks. <br />
<br><br />
<br>'''Concerns Related to the Use of GMO Plants:'''<br />
Although transgenic plants have made a large positive impact on modern agriculture, the use of GMO crops is still a very controversial topic. Issues related to GMOS and health, the environment, economics and ethics are of concern to many people. <br />
<br><br />
*'''Health Concerns''' – While the introduction of genetically engineered traits has made a huge impact on crop yields, many people are concerns about the introduction of transgenes into the human food chain. Although extensive testing of GMO crops is required by the FDA and USDA before approval for cultivation, some people still worry that GMO foods are “unnatural” and may cause health problems when consumed. Many argue that even though health issues have not yet arisen, problems may occur in the future. <br />
<br><br />
*'''Environmental Concerns''' – The widespread use of GMO crops has concerned environmentalist on a number of levels. First of all, many see the use of herbicide resistant crops as being harmful to the environment because farmers will be prone to increase their use of specific herbicide to control weeds. People also worry about the possible transfer of the herbicide resistant genes to closely related weedy plant species could lead to the development of superweeds. Concerns have also arisen from the expression of insecticidal genes that may lead to the killing of not only pest insects, but also beneficial insects such as pollinators or predatory insects. <br />
<br><br />
*'''Economic Concerns''' – Many object to the fact that the distribution of GMO seed is controlled by large multinational conglomerates. And that due to proprietary issues, ownership of the seed, even in later generations, remains the property of these companies. Therefore farmers can no longer save seed from one year’s harvest to plant as next year’s crop. While this has been an issue since the advent of hybrid seeds in the 1950s and 1960s, mainly object to these practices because they prevent small farmers from realizing the benefits of GMOs while at the same time practicing subsistence farming. <br />
<br><br />
*'''Ethical Concerns''' – In regards to people’s personal beliefs, some feel that manipulating plant genomes through recombinant DNA approaches is an abomination to their god. They see this as an unnatural act that goes against the higher order of things. <br />
<br><br />
<br>While many of these concerns have been address through technological developments and stringent government regulations, many people will always reject GMO due to conflicts with basic belief systems. While it is difficult to convince such people that the benefits of GMO plants outweigh the concerns, our job as scientist is to alleviate their concerns with rational science based dialog. <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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/Transgenic_PlantsTeam:Nevada/Transgenic Plants2010-10-28T00:06:55Z<p>Hilarya: /* Transgenic Plants: into the Wild */</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 />
<p>'''Technological Advances from Genetically Engineered Plants'''<br />
<br><br />
Since the initial development of Agrobacterium transformation systems, many plant species including tobacco, tomato, potato, rice, soybean, mint, melon, cucumber, pine and poplar trees, and many others have been transformed using this ingenious bacterial vector. Important traits have been engineered into plants including pest and weed resistance, increased nutritional value, environmental stress tolerance, the production of pharmaceutical and industrial proteins, and the production of bioactive secondary chemical compounds. Our ability to genetically engineer plants has revolutionized agriculture by increasing crop yields while drastically decreasing the application of herbicides and pesticides. This technology is necessary to allow farmers to produce sufficient food for a growing global population. Furthermore, plants are currently being engineered to produce fuel and chemical alternatives to petroleum based products. Because plants are net consumers of atmospheric carbon dioxide, they are currently being seen as a means to sequester greenhouse gases while at the same time replacing petroleum and coal as chemical feedstocks. <br />
<br><br />
<br>'''Concerns Related to the Use of GMO Plants:'''<br />
Although transgenic plants have made a large positive impact on modern agriculture, the use of GMO crops is still a very controversial topic. Issues related to GMOS and health, the environment, economics and ethics are of concern to many people. <br />
<br><br />
<br>*'''Health Concerns''' – While the introduction of genetically engineered traits has made a huge impact on crop yields, many people are concerns about the introduction of transgenes into the human food chain. Although extensive testing of GMO crops is required by the FDA and USDA before approval for cultivation, some people still worry that GMO foods are “unnatural” and may cause health problems when consumed. Many argue that even though health issues have not yet arisen, problems may occur in the future. <br />
<br><br />
<br>*'''Environmental Concerns''' – The widespread use of GMO crops has concerned environmentalist on a number of levels. First of all, many see the use of herbicide resistant crops as being harmful to the environment because farmers will be prone to increase their use of specific herbicide to control weeds. People also worry about the possible transfer of the herbicide resistant genes to closely related weedy plant species could lead to the development of superweeds. Concerns have also arisen from the expression of insecticidal genes that may lead to the killing of not only pest insects, but also beneficial insects such as pollinators or predatory insects. <br />
<br><br />
<br>*'''Economic Concerns''' – Many object to the fact that the distribution of GMO seed is controlled by large multinational conglomerates. And that due to proprietary issues, ownership of the seed, even in later generations, remains the property of these companies. Therefore farmers can no longer save seed from one year’s harvest to plant as next year’s crop. While this has been an issue since the advent of hybrid seeds in the 1950s and 1960s, mainly object to these practices because they prevent small farmers from realizing the benefits of GMOs while at the same time practicing subsistence farming. <br />
<br><br />
<br>*'''Ethical Concerns''' – In regards to people’s personal beliefs, some feel that manipulating plant genomes through recombinant DNA approaches is an abomination to their god. They see this as an unnatural act that goes against the higher order of things. <br />
<br><br />
<br>While many of these concerns have been address through technological developments and stringent government regulations, many people will always reject GMO due to conflicts with basic belief systems. While it is difficult to convince such people that the benefits of GMO plants outweigh the concerns, our job as scientist is to alleviate their concerns with rational science based dialog. <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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/Transgenic_PlantsTeam:Nevada/Transgenic Plants2010-10-27T22:07:28Z<p>Hilarya: /* Transgenic Plants: into the Wild */</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 />
<p>'''Technological Advances from Genetically Engineered Plants'''<br />
<br><br />
Since the initial development of Agrobacterium transformation systems, many plant species including tobacco, tomato, potato, rice, soybean, mint, melon, cucumber, pine and poplar trees, and many others have been transformed using this ingenious bacterial vector. Important traits have been engineered into plants including pest and weed resistance, increased nutritional value, environmental stress tolerance, the production of pharmaceutical and industrial proteins, and the production of bioactive secondary chemical compounds. Our ability to genetically engineer plants has revolutionized agriculture by increasing crop yields while drastically decreasing the application of herbicides and pesticides. This technology is necessary to allow farmers to produce sufficient food for a growing global population. Furthermore, plants are currently being engineered to produce fuel and chemical alternatives to petroleum based products. Because plants are net consumers of atmospheric carbon dioxide, they are currently being seen as a means to sequester greenhouse gases while at the same time replacing petroleum and coal as chemical feedstocks. <br />
<br><br />
<br>However, there has been recent controversy concerning the use of transgenic plants and organisms. These issues include economical, environmental, ethical, and health concerns. We have developed the following powerpoint outlining and discussing a short history as well as some issues concerning GMOs.</p><br />
<br />
https://static.igem.org/mediawiki/2010/4/4d/IGEM_gmo_ppt.pdf<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/ModelingTeam:Nevada/Modeling2010-10-27T21:54:27Z<p>Hilarya: /* Acknowledgments */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Modeling.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
== Modeling ==<br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png<br />
----<br />
=== Introduction ===<br />
<br />
<p>Plant stress responses are often cascades involving hundreds of genes and gene products. The possible interactions in these cascades are astronomical. Therefore, the 2010 Nevada iGEM team worked with Bioinformatics Professor, Karen Schlauch, to use a computational method that could quickly analyze possible transcriptional regulation pathways, using either microarray data or data from continuous fluorometry experiments. In conjunction with the use of tobacco BY-2 (NT1) cells, this method could allow for even greater time efficiency in identifying important aspects of gene networks in plants. The method was intended to allow for easier identification of promoters useful to the team’s objective of creating remote plant biosensors. The method uses a Boolean network approach to examine the gene network and its possible regulatory system. We first viewed transcripts as “on” when above a threshold value and “off” for lesser values. All Boolean networks that could generate our dataset were generated and evaluated. In this manner, we were able to look at all possible interactions between genes based on the Boolean approach.</p><br />
<br />
<br />
=== The DREB1 Pathway ===<br />
<br><br />
<br><br />
<br><br />
<br />
=== Boolean Networks ===<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Data ===<br />
<br />
When it became clear that the team would not have sufficient time to perform fluorometry experiments and analyze data by November, it was decided that microarray experiments published to internet databases would have to do. All data was originally obtained from a 24-hour time course microarray experiment performed by Jian-Kang Zhu, ''et al,'' and published on the Gene Expression Omnibus database (Zhu, ''et al.'' 2005). This allowed for a proof-of-concept to see if the Boolean network would support what was known about the DREB1 pathway from published literature. <br />
<br />
[https://2010.igem.org/Team:Nevada/Original_Data<u>Click here to see the initial data set</u>]<br />
<br />
Because this data consisted of only four time points, all eight genes had similar Boolean values at each time point. Therefore, the Boolean functions for each were essentially the same and numbered in the billions. This provided little data for interpretation. Several methods were used to tease out differences in expression, so that the time courses would be sufficiently different. First, the threshold value for "on" was raised to 2^3.5 rather than 4. Second, The data was interpolated to estimate extra time points within the 24-hour time course. Finally, the number of inputs for each gene was limited to four, as it was deemed unlikely that any gene in this network was receiving input from 5 or more.<br />
<br />
[https://2010.igem.org/Team:Nevada/Interpolated_Data<u>Click here to see the interpolated data set and associated Boolean functions</u>]<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Results ===<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png"><img src="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png" style="float:center;width:500px;margin:10px"></a></html><br />
<br><br />
<br />
=== Acknowledgments ===<br />
<br />
The 2010 Nevada iGEM team would like to thank '''Karen Schlauch''' for all of her hard work, performing computational analysis, explaining the concepts of Boolean networking, and working with the team to find biological meaning in the Boolean output functions.<br />
<br><br />
<br />
=== References ===<br />
<br />
'''Zhu, J-K., Lee, B., Henderson, D.''' (2005) The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1. ''Plant Cell.'' Vol. 17, Issue 11, p3155-3175.<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Template:Nevada_topbarTemplate:Nevada topbar2010-10-27T21:50:14Z<p>Hilarya: </p>
<hr />
<div><html><br />
<div id="headerlinks"><br />
<ul id="nav"><br />
<li><br />
<a class="bannertoplinks" href="https://2010.igem.org/Team:Nevada">Home</a><br />
</li> <br />
<li><br />
<a class="bannertoplinks" href="https://2010.igem.org/Team:Nevada/Team">Team profile</a><br />
</li><br />
<li><br />
<a class="bannertoplinks" href="#">Project</a><br />
<ul style="z-index:1"><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/plants_as_remote_sensors">Remote Sensors</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/long_term_goals">Long Term Goals</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="bannertoplinks" href="#">Parts</a><br />
<ul style="z-index:1"><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/promoters">promoters</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/plant_compatible_reporters">reporters</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/plasmid_w_terminator_sequences">plasmid + term.</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/Composite">composite</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/ccdB">ccdB</a></li><br />
</ul><br />
</li><br />
<li> <br />
<a class="bannertoplinks" href="https://2010.igem.org/Team:Nevada/registry_submissions">Registry Submissions</a><br />
</li><br />
<li><br />
<a class="bannertoplinks" href="https://2010.igem.org/Team:Nevada/Transformations">NT Cell Transformations</a><br />
<ul style="z-index:1"><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/Results" tabindex="2">Results</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/BY-2 (NT1)Transformation Protocol" tabindex="1">Protocol</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/Agrobacterium Transformations" tabindex="3">Agro. Trans.</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/Transgenic Plants" tabindex="3">Transgenic Plants</a></li><br />
</ul> <br />
</li><br />
<li><br />
<a class="bannertoplinks" href="#">Other Stuff</a><br />
<ul style="z-index:1"><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/Modeling">Modeling</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/Safety">Safety</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/Notebook">Notebook</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/fundraising_sponsorships">Fundraising & Sponsorships</a><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Nevada/Team Nevada: Plant Summit">Plant Summit</a></li><br />
<li><a class="bannerlinks" href="http://biotechniques.com/news/iGEM-competitors-gear-up-for-2010-challenge/biotechniques-304538.html">Media</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:Freiburg_Bioware/Team/Cuckoo_Clock">Cuckoo Clock</a></li><br />
<br />
</ul><br />
</li><br />
<br />
</ul> <br />
</div><br />
</html></div>Hilaryahttp://2010.igem.org/Team:Nevada/CD2InducibleTeam:Nevada/CD2Inducible2010-10-27T21:44:22Z<p>Hilarya: /* Promoters */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Picture 13.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 />
<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 />
<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/35STeam:Nevada/35S2010-10-27T21:44:01Z<p>Hilarya: </p>
<hr />
<div>{{Nevada35S}}<br />
<br />
== Promoters ==<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 />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png '''35S Promoter''' [[Team:Nevada/registry submissions]]<br />
----<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/2/2f/Picture_16.png"><img src="https://static.igem.org/mediawiki/2010/2/2f/Picture_16.png" style="float:left;width:200px;margin:10px"></a></html><p>The 35S promoter is frequently used as a constitutive promoter in plant research, primarily Arabidopsis experiments, and has been demonstrated to work in Nicotiana tabacum (Kuluev et al, 2010). This promoter normally drives transcription of the Cauliflower mosaic virus genome and shows no tissue or developmental specificity (Keller et al, 2002). For these reasons, the 2010 Nevada iGEM team modified the 35S promoter to conform to BioBrick standards, providing a reliable constitutive promoter to future iGEM teams wishing to engineer plants.<br />
<br><br />
<br>'''References'''<br />
<br>'''Keller, M., Haas, M., Bureau, M., Geldreich, A. and Yot, P.''' (2002) Cauliflower mosaic virus: still in the news.<br />
Molecular Plant Pathology, 3(6), 419–429.<br />
<br>'''Kuluev, B. R., Knyazev, A. V., Lebedev, P. Ya., Iljassowa, A. A. and Chemeris, A. V.''' (2010) Construction of Hybrid Promoters of Caulimoviruses and Analysis of Their Activity in Transgenic Plants. Russian Journal of Plant Physiology, Vol. 57, No. 4, 582-589.</p><br />
<br />
<br />
<br />
<!--- The Mission, Experiments ---><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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/RD29ATeam:Nevada/RD29A2010-10-27T21:42:02Z<p>Hilarya: </p>
<hr />
<div>{{nevadaRD29A}}<br />
<br />
== Promoters ==<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 />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png '''RD29A Promoter + Strong Plant Kozak (RBS) + RFP''' [[Team:Nevada/registry submissions]]<br />
<br />
----<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/c1/Picture_17.png"><img src="https://static.igem.org/mediawiki/2010/c/c1/Picture_17.png" class="shadow" style="float:left;width:200px;margin:10px"></a><br />
</html><br />
<br />
<p>In our project, we designed the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter and the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter + Strong Plant Kozak (RBS) + RFP. These both came from the synthetic design that contained the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter + Strong Plant Kozak (RBS) + RFP in the pMA vector.<br />
The <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> promoter was isolated by the use of the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> designed primers in PCR. This was blunt end Topo cloned, digested with EcoR 1 and Pst 1 sites, and was then ligated to the pSB1C3 vector.<br />
The design of the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter + Strong Plant Kozak (RBS) + RFP involved the ligation to the pSB1C3 vector using the EcoR 1 and Pst 1 sites. This composite part contains both the promoter and the reporter gene. This functions when the promoter is activated during environmental stress, which would then allow the expression of red fluorescence in plants that can be used as a warning signal to farmers that their plants are under stress.</p><br />
<br />
<br><br />
<br>'''References'''<br />
<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.<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/RD29ATeam:Nevada/RD29A2010-10-27T21:40:24Z<p>Hilarya: /* Promoters */</p>
<hr />
<div>{{nevadaRD29A}}<br />
<br />
<br />
== Promoters ==<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 />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png '''RD29A Promoter + Strong Plant Kozak (RBS) + RFP''' [[Team:Nevada/registry submissions]]<br />
<br />
----<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/c1/Picture_17.png"><img src="https://static.igem.org/mediawiki/2010/c/c1/Picture_17.png" class="shadow" style="float:left;width:200px;margin:10px"></a><br />
</html><br />
<br />
<p>In our project, we designed the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter and the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter + Strong Plant Kozak (RBS) + RFP. These both came from the synthetic design that contained the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter + Strong Plant Kozak (RBS) + RFP in the pMA vector.<br />
The <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> promoter was isolated by the use of the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> designed primers in PCR. This was blunt end Topo cloned, digested with EcoR 1 and Pst 1 sites, and was then ligated to the pSB1C3 vector.<br />
The design of the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter + Strong Plant Kozak (RBS) + RFP involved the ligation to the pSB1C3 vector using the EcoR 1 and Pst 1 sites. This composite part contains both the promoter and the reporter gene. This functions when the promoter is activated during environmental stress, which would then allow the expression of red fluorescence in plants that can be used as a warning signal to farmers that their plants are under stress.</p><br />
<br />
<br><br />
<br>'''References'''<br />
<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.<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/ResultsTeam:Nevada/Results2010-10-27T21:34:16Z<p>Hilarya: /* NT Cell Transformation Results */</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/2010/9/99/Rd29A_results.png"><img src="https://static.igem.org/mediawiki/2010/9/99/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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/ResultsTeam:Nevada/Results2010-10-27T21:33:41Z<p>Hilarya: /* NT Cell Transformation Results */</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/2010/9/99/Rd29A_results.png"><img src="https://static.igem.org/mediawiki/2010/9/99/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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/ResultsTeam:Nevada/Results2010-10-27T21:32:29Z<p>Hilarya: </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/2010/9/99/Rd29A_results.png"><img src="https://static.igem.org/mediawiki/2010/9/99/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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/File:Rd29A_results.pngFile:Rd29A results.png2010-10-27T21:30:39Z<p>Hilarya: </p>
<hr />
<div></div>Hilaryahttp://2010.igem.org/Team:Nevada/ModelingTeam:Nevada/Modeling2010-10-27T19:52:27Z<p>Hilarya: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Modeling.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
== Modeling ==<br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png<br />
----<br />
=== Introduction ===<br />
<br />
<p>Plant stress responses are often cascades involving hundreds of genes and gene products. The possible interactions in these cascades are astronomical. Therefore, the 2010 Nevada iGEM team worked with Bioinformatics Professor, Karen Schlauch, to use a computational method that could quickly analyze possible transcriptional regulation pathways, using either microarray data or data from continuous fluorometry experiments. In conjunction with the use of tobacco BY-2 (NT1) cells, this method could allow for even greater time efficiency in identifying important aspects of gene networks in plants. The method was intended to allow for easier identification of promoters useful to the team’s objective of creating remote plant biosensors. The method uses a Boolean network approach to examine the gene network and its possible regulatory system. We first viewed transcripts as “on” when above a threshold value and “off” for lesser values. All Boolean networks that could generate our dataset were generated and evaluated. In this manner, we were able to look at all possible interactions between genes based on the Boolean approach.</p><br />
<br />
<br />
=== The DREB1 Pathway ===<br />
<br><br />
<br><br />
<br><br />
<br />
=== Boolean Networks ===<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Data ===<br />
<br />
When it became clear that the team would not have sufficient time to perform fluorometry experiments and analyze data by November, it was decided that microarray experiments published to internet databases would have to do. All data was originally obtained from a 24-hour time course microarray experiment performed by Jian-Kang Zhu, ''et al,'' and published on the Gene Expression Omnibus database (Zhu, ''et al.'' 2005). This allowed for a proof-of-concept to see if the Boolean network would support what was known about the DREB1 pathway from published literature. <br />
<br />
[https://2010.igem.org/Team:Nevada/Original_Data<u>Click here to see the initial data set</u>]<br />
<br />
Because this data consisted of only four time points, all eight genes had similar Boolean values at each time point. Therefore, the Boolean functions for each were essentially the same and numbered in the billions. This provided little data for interpretation. Several methods were used to tease out differences in expression, so that the time courses would be sufficiently different. First, the threshold value for "on" was raised to 2^3.5 rather than 4. Second, The data was interpolated to estimate extra time points within the 24-hour time course. Finally, the number of inputs for each gene was limited to four, as it was deemed unlikely that any gene in this network was receiving input from 5 or more.<br />
<br />
[https://2010.igem.org/Team:Nevada/Interpolated_Data<u>Click here to see the interpolated data set and associated Boolean functions</u>]<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Results ===<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png"><img src="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png" style="float:center;width:500px;margin:10px"></a></html><br />
<br><br />
<br />
=== Acknowledgments ===<br />
<br />
The 2010 Nevada iGEM team would like to thank Karen Schlauch for all of her hard work, performing computational analysis, explaining the concepts of Boolean networking, and working with the team to find biological meaning in the Boolean output functions.<br />
<br><br />
<br />
<br />
=== References ===<br />
<br />
'''Zhu, J-K., Lee, B., Henderson, D.''' (2005) The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1. ''Plant Cell.'' Vol. 17, Issue 11, p3155-3175.<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/ModelingTeam:Nevada/Modeling2010-10-27T19:51:59Z<p>Hilarya: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Modeling.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
== Modeling ==<br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png<br />
----<br />
=== Introduction ===<br />
<br />
<p>Plant stress responses are often cascades involving hundreds of genes and gene products. The possible interactions in these cascades are astronomical. Therefore, the 2010 Nevada iGEM team worked with Bioinformatics Professor, Karen Schlauch, to use a computational method that could quickly analyze possible transcriptional regulation pathways, using either microarray data or data from continuous fluorometry experiments. In conjunction with the use of tobacco BY-2 (NT1) cells, this method could allow for even greater time efficiency in identifying important aspects of gene networks in plants. The method was intended to allow for easier identification of promoters useful to the team’s objective of creating remote plant biosensors. The method uses a Boolean network approach to examine the gene network and its possible regulatory system. We first viewed transcripts as “on” when above a threshold value and “off” for lesser values. All Boolean networks that could generate our dataset were generated and evaluated. In this manner, we were able to look at all possible interactions between genes based on the Boolean approach.</p><br />
<br />
<br />
=== The DREB1 Pathway ===<br />
<br><br />
<br><br />
<br><br />
<br />
=== Boolean Networks ===<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Data ===<br />
<br />
When it became clear that the team would not have sufficient time to perform fluorometry experiments and analyze data by November, it was decided that microarray experiments published to internet databases would have to do. All data was originally obtained from a 24-hour time course microarray experiment performed by Jian-Kang Zhu, ''et al,'' and published on the Gene Expression Omnibus database (Zhu, ''et al.'' 2005). This allowed for a proof-of-concept to see if the Boolean network would support what was known about the DREB1 pathway from published literature. <br />
<br />
[https://2010.igem.org/Team:Nevada/Original_Data<u>Click here to see the initial data set</u>]<br />
<br />
Because this data consisted of only four time points, all eight genes had similar Boolean values at each time point. Therefore, the Boolean functions for each were essentially the same and numbered in the billions. This provided little data for interpretation. Several methods were used to tease out differences in expression, so that the time courses would be sufficiently different. First, the threshold value for "on" was raised to 2^3.5 rather than 4. Second, The data was interpolated to estimate extra time points within the 24-hour time course. Finally, the number of inputs for each gene was limited to four, as it was deemed unlikely that any gene in this network was receiving input from 5 or more.<br />
<br />
[https://2010.igem.org/Team:Nevada/Interpolated_Data<u>Click here to see the interpolated data set and associated Boolean functions</u>]<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Results ===<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png"><img src="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png" style="float:center;width:500px;margin:10px"></a></html><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<br />
=== Acknowledgments ===<br />
<br />
The 2010 Nevada iGEM team would like to thank Karen Schlauch for all of her hard work, performing computational analysis, explaining the concepts of Boolean networking, and working with the team to find biological meaning in the Boolean output functions.<br />
<br><br />
<br />
<br />
=== References ===<br />
<br />
'''Zhu, J-K., Lee, B., Henderson, D.''' (2005) The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1. ''Plant Cell.'' Vol. 17, Issue 11, p3155-3175.<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/ModelingTeam:Nevada/Modeling2010-10-27T19:51:28Z<p>Hilarya: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Modeling.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
== Modeling ==<br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png<br />
----<br />
=== Introduction ===<br />
<br />
<p>Plant stress responses are often cascades involving hundreds of genes and gene products. The possible interactions in these cascades are astronomical. Therefore, the 2010 Nevada iGEM team worked with Bioinformatics Professor, Karen Schlauch, to use a computational method that could quickly analyze possible transcriptional regulation pathways, using either microarray data or data from continuous fluorometry experiments. In conjunction with the use of tobacco BY-2 (NT1) cells, this method could allow for even greater time efficiency in identifying important aspects of gene networks in plants. The method was intended to allow for easier identification of promoters useful to the team’s objective of creating remote plant biosensors. The method uses a Boolean network approach to examine the gene network and its possible regulatory system. We first viewed transcripts as “on” when above a threshold value and “off” for lesser values. All Boolean networks that could generate our dataset were generated and evaluated. In this manner, we were able to look at all possible interactions between genes based on the Boolean approach.</p><br />
<br />
<br />
=== The DREB1 Pathway ===<br />
<br><br />
<br><br />
<br><br />
<br />
=== Boolean Networks ===<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Data ===<br />
<br />
When it became clear that the team would not have sufficient time to perform fluorometry experiments and analyze data by November, it was decided that microarray experiments published to internet databases would have to do. All data was originally obtained from a 24-hour time course microarray experiment performed by Jian-Kang Zhu, ''et al,'' and published on the Gene Expression Omnibus database (Zhu, ''et al.'' 2005). This allowed for a proof-of-concept to see if the Boolean network would support what was known about the DREB1 pathway from published literature. <br />
<br />
[https://2010.igem.org/Team:Nevada/Original_Data<u>Click here to see the initial data set</u>]<br />
<br />
Because this data consisted of only four time points, all eight genes had similar Boolean values at each time point. Therefore, the Boolean functions for each were essentially the same and numbered in the billions. This provided little data for interpretation. Several methods were used to tease out differences in expression, so that the time courses would be sufficiently different. First, the threshold value for "on" was raised to 2^3.5 rather than 4. Second, The data was interpolated to estimate extra time points within the 24-hour time course. Finally, the number of inputs for each gene was limited to four, as it was deemed unlikely that any gene in this network was receiving input from 5 or more.<br />
<br />
[https://2010.igem.org/Team:Nevada/Interpolated_Data<u>Click here to see the interpolated data set and associated Boolean functions</u>]<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Results ===<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png"><img src="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png" style="float:left;width:500px;margin:10px"></a></html><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== Acknowledgments ===<br />
<br />
The 2010 Nevada iGEM team would like to thank Karen Schlauch for all of her hard work, performing computational analysis, explaining the concepts of Boolean networking, and working with the team to find biological meaning in the Boolean output functions.<br />
<br><br />
<br />
<br />
=== References ===<br />
<br />
'''Zhu, J-K., Lee, B., Henderson, D.''' (2005) The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1. ''Plant Cell.'' Vol. 17, Issue 11, p3155-3175.<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/ModelingTeam:Nevada/Modeling2010-10-27T19:50:58Z<p>Hilarya: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Modeling.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
== Modeling ==<br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png<br />
----<br />
=== Introduction ===<br />
<br />
<p>Plant stress responses are often cascades involving hundreds of genes and gene products. The possible interactions in these cascades are astronomical. Therefore, the 2010 Nevada iGEM team worked with Bioinformatics Professor, Karen Schlauch, to use a computational method that could quickly analyze possible transcriptional regulation pathways, using either microarray data or data from continuous fluorometry experiments. In conjunction with the use of tobacco BY-2 (NT1) cells, this method could allow for even greater time efficiency in identifying important aspects of gene networks in plants. The method was intended to allow for easier identification of promoters useful to the team’s objective of creating remote plant biosensors. The method uses a Boolean network approach to examine the gene network and its possible regulatory system. We first viewed transcripts as “on” when above a threshold value and “off” for lesser values. All Boolean networks that could generate our dataset were generated and evaluated. In this manner, we were able to look at all possible interactions between genes based on the Boolean approach.</p><br />
<br />
<br />
=== The DREB1 Pathway ===<br />
<br><br />
<br><br />
<br><br />
<br />
=== Boolean Networks ===<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Data ===<br />
<br />
When it became clear that the team would not have sufficient time to perform fluorometry experiments and analyze data by November, it was decided that microarray experiments published to internet databases would have to do. All data was originally obtained from a 24-hour time course microarray experiment performed by Jian-Kang Zhu, ''et al,'' and published on the Gene Expression Omnibus database (Zhu, ''et al.'' 2005). This allowed for a proof-of-concept to see if the Boolean network would support what was known about the DREB1 pathway from published literature. <br />
<br />
[https://2010.igem.org/Team:Nevada/Original_Data<u>Click here to see the initial data set</u>]<br />
<br />
Because this data consisted of only four time points, all eight genes had similar Boolean values at each time point. Therefore, the Boolean functions for each were essentially the same and numbered in the billions. This provided little data for interpretation. Several methods were used to tease out differences in expression, so that the time courses would be sufficiently different. First, the threshold value for "on" was raised to 2^3.5 rather than 4. Second, The data was interpolated to estimate extra time points within the 24-hour time course. Finally, the number of inputs for each gene was limited to four, as it was deemed unlikely that any gene in this network was receiving input from 5 or more.<br />
<br />
[https://2010.igem.org/Team:Nevada/Interpolated_Data<u>Click here to see the interpolated data set and associated Boolean functions</u>]<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Results ===<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png"><img src="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png" style="float:left;width:500px;margin:10px"></a></html><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<br />
=== Acknowledgments ===<br />
<br />
The 2010 Nevada iGEM team would like to thank Karen Schlauch for all of her hard work, performing computational analysis, explaining the concepts of Boolean networking, and working with the team to find biological meaning in the Boolean output functions.<br />
<br><br />
<br />
<br />
=== References ===<br />
<br />
'''Zhu, J-K., Lee, B., Henderson, D.''' (2005) The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1. ''Plant Cell.'' Vol. 17, Issue 11, p3155-3175.<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/ModelingTeam:Nevada/Modeling2010-10-27T19:49:31Z<p>Hilarya: /* Results */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Modeling.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
== Modeling ==<br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png<br />
----<br />
=== Introduction ===<br />
<br />
<p>Plant stress responses are often cascades involving hundreds of genes and gene products. The possible interactions in these cascades are astronomical. Therefore, the 2010 Nevada iGEM team worked with Bioinformatics Professor, Karen Schlauch, to use a computational method that could quickly analyze possible transcriptional regulation pathways, using either microarray data or data from continuous fluorometry experiments. In conjunction with the use of tobacco BY-2 (NT1) cells, this method could allow for even greater time efficiency in identifying important aspects of gene networks in plants. The method was intended to allow for easier identification of promoters useful to the team’s objective of creating remote plant biosensors. The method uses a Boolean network approach to examine the gene network and its possible regulatory system. We first viewed transcripts as “on” when above a threshold value and “off” for lesser values. All Boolean networks that could generate our dataset were generated and evaluated. In this manner, we were able to look at all possible interactions between genes based on the Boolean approach.</p><br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
=== The DREB1 Pathway ===<br />
<br><br />
<br><br />
<br><br />
<br />
=== Boolean Networks ===<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Data ===<br />
<br />
When it became clear that the team would not have sufficient time to perform fluorometry experiments and analyze data by November, it was decided that microarray experiments published to internet databases would have to do. All data was originally obtained from a 24-hour time course microarray experiment performed by Jian-Kang Zhu, ''et al,'' and published on the Gene Expression Omnibus database (Zhu, ''et al.'' 2005). This allowed for a proof-of-concept to see if the Boolean network would support what was known about the DREB1 pathway from published literature. <br />
<br />
[https://2010.igem.org/Team:Nevada/Original_Data<u>Click here to see the initial data set</u>]<br />
<br />
Because this data consisted of only four time points, all eight genes had similar Boolean values at each time point. Therefore, the Boolean functions for each were essentially the same and numbered in the billions. This provided little data for interpretation. Several methods were used to tease out differences in expression, so that the time courses would be sufficiently different. First, the threshold value for "on" was raised to 2^3.5 rather than 4. Second, The data was interpolated to estimate extra time points within the 24-hour time course. Finally, the number of inputs for each gene was limited to four, as it was deemed unlikely that any gene in this network was receiving input from 5 or more.<br />
<br />
[https://2010.igem.org/Team:Nevada/Interpolated_Data<u>Click here to see the interpolated data set and associated Boolean functions</u>]<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Results ===<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png"><img src="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png" style="float:left;width:500px;margin:10px"></a></html><br />
<br />
=== Acknowledgments ===<br />
<br />
The 2010 Nevada iGEM team would like to thank Karen Schlauch for all of her hard work, performing computational analysis, explaining the concepts of Boolean networking, and working with the team to find biological meaning in the Boolean output functions.<br />
<br><br />
<br><br />
<br><br />
<br />
=== References ===<br />
<br />
Zhu, J-K., Lee, B., Henderson, D. (2005) The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1. ''Plant Cell.'' Vol. 17, Issue 11, p3155-3175.<br />
<br />
<br />
<br />
<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/Original_DataTeam:Nevada/Original Data2010-10-27T19:43:40Z<p>Hilarya: /* References */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Binary_Bender.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
<br />
<br />
== Original Data Set ==<br />
<br />
Data collected from microarray experiments performed by Jian-Kang Zhu, ''et al,'' and published on the Gene Expression Omnibus database (Zhu, ''et al.'' 2005). <br />
<br />
{| border="3"<br />
|Raw Data <br />
|ICE1 <br />
|DREB1A <br />
|DREB1B <br />
|DREB1C <br />
|RD29A <br />
|ZAT6 <br />
|RAP2.1 <br />
|RAP2.6<br />
|-<br />
|t0 <br />
|1358.1 <br />
|96.6 <br />
|24.5 <br />
|69.4 <br />
|1509.2 <br />
|145.3 <br />
|100.4 <br />
|58.5<br />
|-<br />
|t1 <br />
|1487.7 <br />
|6041 <br />
|4826.5 <br />
|11809.1 <br />
|3284.5 <br />
|676.3 <br />
|103.4 <br />
|57.3<br />
|-<br />
|t2 <br />
|1547.9 <br />
|6181.2 <br />
|4099 <br />
|11249.3 <br />
|10644.4 <br />
|1620.6 <br />
|357.8 <br />
|52.3<br />
|-<br />
|t3 <br />
|1476.5 <br />
|1452.8 <br />
|815.4 <br />
|4510.4 <br />
|18745.9 <br />
|2850.7 <br />
|1599.3 <br />
|125<br />
|}<br />
<br><br />
<br><br />
<br><br />
<br />
== Log Base 2 of Normalized Ratios ==<br />
<br />
Values were normalized to 1 at t0, and log base 2 values were taken.<br />
<br />
{| border="3"<br />
|Log2 Ratios <br />
|ICE1 <br />
|DREB1A <br />
|DREB1B <br />
|DREB1C <br />
|RD29A <br />
|ZAT6 <br />
|RAP2.1 <br />
|RAP2.6<br />
|-<br />
|t0 <br />
|0 <br />
|0 <br />
|0 <br />
|0 <br />
|0 <br />
|0 <br />
|0 <br />
|0<br />
|-<br />
|t1 <br />
|0.1315 <br />
|5.9666 <br />
|7.6221 <br />
|7.4107 <br />
|1.1219 <br />
|2.2186 <br />
|0.0425 <br />
| -0.0299<br />
|-<br />
|t2 <br />
|0.1887 <br />
|5.9997 <br />
|7.3863 <br />
|7.3407 <br />
|2.8182 <br />
|3.4794 <br />
|1.8334 <br />
| -0.1616<br />
|-<br />
|t3 <br />
|0.1206 <br />
|3.9107 <br />
|5.0567 <br />
|6.0222 <br />
|3.6347 <br />
|4.2942 <br />
|3.9936 <br />
|1.0954<br />
|}<br />
<br><br />
<br><br />
<br><br />
<br />
== Boolean Values (Threshold of 2.0) ==<br />
<br />
Values from the log base 2 table which were greater than or equal to a 2.0-fold increase were assigned values of 1 ( "on" ) and values under 3.5 were assigned values of 0 ( "off" ). <br />
<br />
{| border="3"<br />
|Boolean Values <br />
|ICE1 <br />
|DREB1A <br />
|DREB1B <br />
|DREB1C <br />
|rd29A <br />
|ZAT6 <br />
|RAP2.1 <br />
|RAP2.6<br />
|-<br />
|t0 <br />
|0 <br />
|0 <br />
|0 <br />
|0 <br />
|0 <br />
|0 <br />
|0 <br />
|0<br />
|-<br />
|t1 <br />
|0 <br />
|1 <br />
|1 <br />
|1 <br />
|0 <br />
|1 <br />
|0 <br />
|0<br />
|-<br />
|t2 <br />
|0 <br />
|1 <br />
|1 <br />
|1 <br />
|1 <br />
|1 <br />
|0 <br />
|0<br />
|-<br />
|t3 <br />
|0 <br />
|1 <br />
|1 <br />
|1 <br />
|1 <br />
|1 <br />
|1 <br />
|0<br />
|}<br />
<br><br />
<br><br />
<br><br />
<br />
== Boolean Input Functions ==<br />
<br />
The number of Boolean inputs for this data amounted to 20 megabytes worth of text files, and thus, has been omitted. The extremely large number of inputs going into each gene indicated that the process needed some refinement to limit the number of possible interactions between these genes. This lead to the data interpolation and limiting the Boolean inputs to being no greater than 4 for each gene.<br />
<br />
For some examples of Boolean Input Functions, please see the [https://2010.igem.org/Team:Nevada/Interpolated_Data<u>interpolated data page.</u>]<br />
<br><br />
<br><br />
<br><br />
<br />
== References ==<br />
<br />
'''Zhu, J-K., Lee, B., Henderson, D.''' (2005) The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1. ''Plant Cell.'' Vol. 17, Issue 11, p3155-3175.</div>Hilaryahttp://2010.igem.org/Team:Nevada/Agrobacterium_TransformationsTeam:Nevada/Agrobacterium Transformations2010-10-27T19:38:39Z<p>Hilarya: /* How Agrobacterium Transforms Plants */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Agro header.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<br />
== How Agrobacterium Transforms Plants ==<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 />
'''Agrobacterium Transformation of Plants'''<br />
<br><br />
Agrobacterium mediated transformation is the most widely used means of integrating DNA fragments of interested into the genome of plant cells. This process takes advantage of the naturally occurring plant pathogen, ''Agrobacterium tumefaciens''. This soil bacterium is the causative agent responsible for crown gall disease in a wide range of plant species. Pathogenic Agrobacterium carry a large plasmid, referred to as the Tumor Inducing or Ti plasmid, which is required for disease transmission. The Ti plasmid contains virulence genes (vir), which code for the protein machinery required for transfer and integration of disease causing genes into the host plants genome. The Ti plasmid also contains a set of genes designated as the T-DNA. The T-DNA is flanked by inverted repeat sequences referred to as the Left Border (LB) and Right Border (RB) sequences. Any DNA sequences situated between the LB and RB will be inserted into the plant host genome. In pathogenic strains of ''A. tumefaciens'', the border sequences flank genes involved in tumor formation in infected plants. Once integrated into the host genome, these genes, referred to as the oncogenes, hijack the host plant to produce the protein machinery needed for the production of phytohormones. These phytohormones cause uncontrolled cell proliferation which resulting in the formation of a gall or tumor. In addition to the oncogenes, the T-DNA region also encoded biosynthetic proteins for the production of unusual amino acids referred to as opines. Therefore, by integrating the T-DNA into the host, the bacteria forces the plant host to create both a habitat and a carbon/nitrogen source on which the bacteria could survive. <br />
<br><br />
<br>'''Development of Agrobacterium as a Vector for Plant Genetic Engineering'''<br />
<br><br />
In the 1980’s scientist took advantage of ''A. tumefacien’s'' ability to insert foreign DNA into plant chromosomes to develop highly efficient vectors for plant transformation and genetic engineering. By swapping the oncogenes from the T-DNA region of the Ti plasmid with any plant gene expression cassette (containing a plant promoter, a gene of interest and a transcriptional terminator sequence), they were able to integrate their particular gene of interest into the host genome without causing the disease. They later found that the vir gene products could function in trans such that the T-DNA present on a separate plasmid from the plasmid carrying the vir genes could still be transferred to the host plant. This system, referred to as the binary transformation system, was composed of an Agrobacterium strain containing a Ti plasmid in which the T-DNA was deleted and a second plasmid, which carries the LB and RB sequences flanking two plant gene expression cassettes. One of these cassettes was used to express a gene of interest and the second was used to express an antibiotic resistance gene for the selection of positive transformation events. <br />
<br />
<br><br />
'''Molecular Progression of Agrobacterium Infection'''<br />
<br>1. Chemical perception of host<br />
<br>2. Induction of virulence genes<br />
<br>3. Formation of infection apparatus<br />
<br>4. Preparation of T-DNA<br />
<br>5. Export T-DNA to plant cell<br />
<br>6. Import T-DNA to plant nucleus<br />
<br>7. Integration of T-DNA into host genome<br />
<br>8. Expression of T-DNA <br />
<br><br />
<br><br />
'''Agrobacterium Transformation "Up Close":'''<br />
<br><br />
<html><a href="https://static.igem.org/mediawiki/2010/b/be/Up_close_Agro.png"><img src="https://static.igem.org/mediawiki/2010/b/be/Up_close_Agro.png" class="shadow" style="float:center;width:500px;margin:10px"></a><br />
</html><br />
<br><br />
<br><br />
'''Agrobacterium Transformation Overview:'''<br />
<html><a href="https://static.igem.org/mediawiki/2010/d/d0/Agro_theory.png"><img src="https://static.igem.org/mediawiki/2010/d/d0/Agro_theory.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
</html></div>Hilaryahttp://2010.igem.org/Team:Nevada/BY-2_(NT1)Transformation_ProtocolTeam:Nevada/BY-2 (NT1)Transformation Protocol2010-10-27T19:36:35Z<p>Hilarya: /* BY-2 (NT1) Cell Transformation Protocol */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:NT cell transformations.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<br />
== BY-2 (NT1) Cell Transformation Protocol ==<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: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 too 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 subcultured 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 />
<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/plasmid_w_terminator_sequencesTeam:Nevada/plasmid w terminator sequences2010-10-27T19:33:52Z<p>Hilarya: /* Plasmid with Terminator Sequence */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Final Plasmid header.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
== Plasmid with Terminator Sequence ==<br />
<br />
<p>'''piGEM10 Plasmid Construction'''<br />
<br>Plant transformation vector piGEM10 was derived from the vector pBIB HYG (Becker (1990) Nuc Acid Res 18: 203). To construct this plasmid, a synthetic gene containing the Arabidopsis stress induced RD29 promoter, a red fluorescent protein (RFP) containing a plant optimized ribosome binding site, and the nopoline synthase (NOS) 3’ plant specific transcriptional terminator. The NOS terminator contains a plant specific sequences that signals transcriptional termination and the addition of a polyadenyled 3’ tail to the mature mRNA. HinD3, EcoR1 and Xba1 restriction sites were added respectively to the 5’ end of the RD29 promoter. Spe1 and Pst1 sites were inserted between the stop codon of the RFP gene and the NOS terminator. Additionally, an Mfe1 site was added to the 3’ end of the NOS 3’ terminator. This synthetic gene was subcloned into a plasmid designated as piGEM Nevada.</p><br />
<br><br />
<p>The synthetic gene construct from piGEM Nevada was then subcloned as a HinD3 Mfe1 fragment into the HinD3 EcoR1 site of pBIB HYG. Because Mfe1 and EcoR1 restriction sites generate compatible sticky ends that when joined cannot be re-cleaved by either enzyme, by subcloning the synthetic gene as a HinD3 Mfe1 fragment instead of a HinD3 EcoR1 fragment we were able to destroy the EcoR1 site located at the 3’ end of the NOS 3’ terminator. The resulting plasmid now contains Biobrick compatible cut sites flanking the 5’ end of the promoter and 3’ end of the reporter gene. This configuration will allow for the exchange of the RD29 promoter / RFP fusion with any other promoter / gene fusion construct. We are in the process of removing an unwanted Pst1 site located in the plasmid backbone. However, the vector is still useable now by cloning promoter / gene fusions as EcoR1 Spe1 fragments into the EcoR1 Spe1 sites of piGEM10.</p><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/0/05/PiGEM10.gif"><img src="https://static.igem.org/mediawiki/2010/0/05/PiGEM10.gif" class="shadow" style="float:left;width:900px;margin:10px"></a></html> <br />
<br><br />
<br><p>pBIB HYG was chosen as the basis for our vector construction based on the following criteria. First, unlike many plant transformation vectors, pBIB HYG appears to be freely available in the public domain. Patent searches failed to turn up any past or pending patents that may restrict the use of the plasmid. Second, pBIB HYG is derived from the well characterized pBIN19 plant transformation vector series, which has been used in the transformation of a wide variety of plant species. Third, pBIB HYG required the fewest modifications to become compatible with current Biobrick standards.<br />
<br><br />
<br>'''piGEM10 Features'''<br />
<br>The piGEM10 backbone, which is derived from pBIN19, possesses a RK2 broad host plasmid origin of replication that allows for its use in both E. coli and Agrobacterium tumefaciens. The RK2 origin of replication allows the investigator to manipulate the plasmid in E. coli and then transform the finished plasmid to the appropriate Agrobacterium strain for transformation into plants. One unfortunate consequence of plasmids with RK2 origins is that they are low copy number plasmids and therefore one must use larger cultures when isolating these plasmids. piGEM10 also possesses a bacterial expressed neomycin phosphotransferase 2 (NPT2) gene that confers kanamycin resistance in bacteria carrying this plasmid. 50 μg/mL of kanamycin should be used to select for bacterial transformants containing this plasmid. The plasmid also contains the T-DNA Left (LB) and Right Border (RB) sequences, which are required for the integration of recombinant DNA fragments into the genome of target plant transformation host. Between the LB and RB are two plant gene expression cassettes. The first expression cassette consist of the nopoline synthase promoter, the hygromycin B phosphotransferase (HPT) gene and a plant polyadenylation signal sequences. The nopoline synthase promoter allows for high-level and constitutive transcription HPT in plant cells. When selecting for plants transformed with this vector, 50 μg/mL hygromycin should be added to the tissue culture medium. The second gene cassette consists of the plant stress inducible RD29 promoter; a plant optimized red fluorescent protein gene and nopoline synthase NOS terminator.</p><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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/plant_compatible_reportersTeam:Nevada/plant compatible reporters2010-10-27T19:32:30Z<p>Hilarya: /* Reporters */</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 />
<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. A 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><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><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><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 />
{| 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>Hilaryahttp://2010.igem.org/Team:Nevada/plant_compatible_reportersTeam:Nevada/plant compatible reporters2010-10-27T19:30:41Z<p>Hilarya: /* Reporters */</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 />
<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><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><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><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 />
{| 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>Hilaryahttp://2010.igem.org/Team:Nevada/ccdBTeam:Nevada/ccdB2010-10-27T19:29:02Z<p>Hilarya: /* ccdB */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Ccdb Header new.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<br />
<p>&nbsp;</p><br />
<br />
== 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 />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
----<br />
<br />
<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 was 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 (Left). <br />
<br><br />
*PCR gel confirming the presence of ccdB in amplified sample (Right). <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 />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
<br />
<br />
<br />
<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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/plants_as_remote_sensorsTeam:Nevada/plants as remote sensors2010-10-27T19:27:34Z<p>Hilarya: /* Plants as Remote Sensors */</p>
<hr />
<div>{{Nevada_css}}<br />
<html><br />
<div id="allcontent"><br />
<img class="bannerimage" src="https://static.igem.org/mediawiki/2010/1/1b/Managed-canopy-sm.jpg" width="950px" height="266"><br /><br />
</html><br />
{{Nevada_topbar}}<br />
<br />
==Plants as Remote Sensors==<br />
<br />
<p>The passage of “The Stockholm Convention on Persistant Organic Pollutants” in 2008 clearly demonstrates that the world has taken a much more active approach in addressing concerns about the environmental and ecological impact of pollutants (Rodriguez-Mozaz et al, 2006). By demonstrating the practicality of plants as model biosensors for remote sensing, the Nevada team has addresses several issues that have plagued the field of biosensors since their original conception. First by utilizing a natural receptor that can be localized to a specific cell type in plants, Nevada has shown the usefulness of transgenic plant biosensors that will respond locally to toxic compounds. In addition, natural biosensors that do not inhibit basic metabolic functions and only fluoresce allow for a simpler model system that is easily adapted for future uses. Secondly, model plant organisms are much larger reporters of environmental stresses and can be easily clustered together to form ‘reporter clusters’ that are easily viewed at the source of the contamination. Finally, the natural life cycle of plant organisms (when compared to traditional microorganism biosensors) is much longer and allows for the continued monitoring of a contaminated area over a much longer time while still providing a feasible option to control the plant’s reproductive cycle. As discussed in “Perspective on Optical Biosensors and Integrated Sensor Systems” (Ligler, 2009), the use of on-site optical biosensors to tackle complex and difficult issues like environmental monitoring, food testing, and even counter- terrorism are driving issues in the proliferation of remote sensing technology.</p><br />
<br><br />
<p>Despite these potential successes, challenges still remain when utilizing and applying biosensors to environmental concerns. Societal concerns about contamination with wild type plants are an ongoing and legitimate concern. It may be possible to eliminate such concerns by engineering in a sterility defect that is coupled with a fluorescent reporter; it may be possible to prevent transgenic contamination. For example, utilizing satellites to image biosensors from space would require a different, possibly IR receptor that can be viewed at great distances. Despite concerns for practicality and issues of feasibility, by simply demonstrating that biosensors in plants is possible, further research is required to determine how advantageous plant biosensors are when weighing the concerns for successfully remediating the environment.<br />
<br><br />
<br>'''References'''<br />
<br>'''Rodriguez-Mozaz et al.''', 2006, Analytical. Bioanal Chem. 386: 1025-1041<br />
<br>'''Ligler, F.S.''', 2009, Analytical. Chem. 81: 519-526</p><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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/Transgenic_PlantsTeam:Nevada/Transgenic Plants2010-10-27T19:26:35Z<p>Hilarya: /* Transgenic Plants: into the Wild */</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 />
<p>'''Technological Advances from Genetically Engineered Plants'''<br />
<br><br />
Since the initial development of Agrobacterium transformation systems, many plant species including tobacco, tomato, potato, rice, soybean, mint, melon, cucumber, pine and poplar trees, and many others have been transformed using this ingenious bacterial vector. Important traits have been engineered into plants including pest and weed resistance, increased nutritional value, environmental stress tolerance, the production of pharmaceutical and industrial proteins, and the production of bioactive secondary chemical compounds. Our ability to genetically engineer plants has revolutionized agriculture by increasing crop yields while drastically decreasing the application of herbicides and pesticides. This technology is necessary to allow farmers to produce sufficient food for a growing global population. Furthermore, plants are currently being engineered to produce fuel and chemical alternatives to petroleum based products. Because plants are net consumers of atmospheric carbon dioxide, they are currently being seen as a means to sequester greenhouse gases while at the same time replacing petroleum and coal as chemical feedstocks. <br />
<br><br />
<br>However, there has been recent controversy concerning the use of transgenic plants and organisms. These issues include economical, environmental, ethical, and health concerns. We have developed the following graphics outlining and discussing a short history as well as some issues concerning GMOs.</p><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/d/d9/GMOs_1.png"><img src="https://static.igem.org/mediawiki/2010/d/d9/GMOs_1.png" class="shadow" style="float:center;width:800px;margin:10px"></a><br />
</html><br />
<html><a href="https://static.igem.org/mediawiki/2010/e/ed/GMOs_2.png"><img src="https://static.igem.org/mediawiki/2010/e/ed/GMOs_2.png" class="shadow" style="float:center;width:800px;margin:10px"></a><br />
</html><br />
<html><a href="https://static.igem.org/mediawiki/2010/6/61/GMOS_3.png"><img src="https://static.igem.org/mediawiki/2010/6/61/GMOS_3.png" class="shadow" style="float:center;width:800px;margin:10px"></a><br />
</html><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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/Transgenic_PlantsTeam:Nevada/Transgenic Plants2010-10-27T19:25:59Z<p>Hilarya: /* Transgenic Plants: into the Wild */</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 />
<p>'''Technological Advances from Genetically Engineered Plants'''<br />
<br><br />
Since the initial development of Agrobacterium transformation systems, many plant species including tobacco, tomato, potato, rice, soybean, mint, melon, cucumber, pine and poplar trees, and many others have been transformed using this ingenious bacterial vector. Important traits have been engineered into plants including pest and weed resistance, increased nutritional value, environmental stress tolerance, the production of pharmaceutical and industrial proteins, and the production of bioactive secondary chemical compounds. Our ability to genetically engineer plants has revolutionized agriculture by increasing crop yields while drastically decreasing the application of herbicides and pesticides. This technology is necessary to allow farmers to produce sufficient food for a growing global population. Furthermore, plants are currently being engineered to produce fuel and chemical alternatives to petroleum based products. Because plants are net consumers of atmospheric carbon dioxide, they are currently being seen as a means to sequester greenhouse gases while at the same time replacing petroleum and coal as chemical feedstocks. <br />
<br><br />
<br>However, there has been recent controversy concerning the use of transgenic plants and organisms. These issues include economical, environmental, ethical, and health concerns. We have developed the following graphics outlining and discussing a short history as well as some issues concerning GMOs. This images has been made available to help educate and for use by other participants.</p><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/d/d9/GMOs_1.png"><img src="https://static.igem.org/mediawiki/2010/d/d9/GMOs_1.png" class="shadow" style="float:center;width:800px;margin:10px"></a><br />
</html><br />
<html><a href="https://static.igem.org/mediawiki/2010/e/ed/GMOs_2.png"><img src="https://static.igem.org/mediawiki/2010/e/ed/GMOs_2.png" class="shadow" style="float:center;width:800px;margin:10px"></a><br />
</html><br />
<html><a href="https://static.igem.org/mediawiki/2010/6/61/GMOS_3.png"><img src="https://static.igem.org/mediawiki/2010/6/61/GMOS_3.png" class="shadow" style="float:center;width:800px;margin:10px"></a><br />
</html><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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/Transgenic_PlantsTeam:Nevada/Transgenic Plants2010-10-27T19:25:13Z<p>Hilarya: /* Transgenic Plants: into the Wild */</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 />
<p>'''Technological Advances from Genetically Engineered Plants'''<br />
<br><br />
Since the initial development of Agrobacterium transformation systems, many plant species including tobacco, tomato, potato, rice, soybean, mint, melon, cucumber, pine and poplar trees, and many others have been transformed using this ingenious bacterial vector. Important traits have been engineered into plants including pest and weed resistance, increased nutritional value, environmental stress tolerance, the production of pharmaceutical and industrial proteins, and the production of bioactive secondary chemical compounds. Our ability to genetically engineer plants has revolutionized agriculture by increasing crop yields while drastically decreasing the application of herbicides and pesticides. This technology is necessary to allow farmers to produce sufficient food for a growing global population. Furthermore, plants are currently being engineered to produce fuel and chemical alternatives to petroleum based products. Because plants are net consumers of atmospheric carbon dioxide, they are currently being seen as a means to sequester greenhouse gases while at the same time replacing petroleum and coal as chemical feedstocks. <br />
<br><br />
<br>However, there has been recent controversy concerning the use of transgenic plants and organisms. These issues include economical, environmental, ethical, and health concerns. We have developed the following graphics outlining and discussing a short history and issues concerning GMOs. This images has been made available to help educate and for use by other participants.</p><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/d/d9/GMOs_1.png"><img src="https://static.igem.org/mediawiki/2010/d/d9/GMOs_1.png" class="shadow" style="float:center;width:800px;margin:10px"></a><br />
</html><br />
<html><a href="https://static.igem.org/mediawiki/2010/e/ed/GMOs_2.png"><img src="https://static.igem.org/mediawiki/2010/e/ed/GMOs_2.png" class="shadow" style="float:center;width:800px;margin:10px"></a><br />
</html><br />
<html><a href="https://static.igem.org/mediawiki/2010/6/61/GMOS_3.png"><img src="https://static.igem.org/mediawiki/2010/6/61/GMOS_3.png" class="shadow" style="float:center;width:800px;margin:10px"></a><br />
</html><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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/Team:Nevada/Transgenic_PlantsTeam:Nevada/Transgenic Plants2010-10-27T19:24:42Z<p>Hilarya: /* Transgenic Plants: into the Wild */</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 />
<p>'''Technological Advances from Genetically Engineered Plants'''<br />
<br><br />
Since the initial development of Agrobacterium transformation systems, many plant species including tobacco, tomato, potato, rice, soybean, mint, melon, cucumber, pine and poplar trees, and many others have been transformed using this ingenious bacterial vector. Important traits have been engineered into plants including pest and weed resistance, increased nutritional value, environmental stress tolerance, the production of pharmaceutical and industrial proteins, and the production of bioactive secondary chemical compounds. Our ability to genetically engineer plants has revolutionized agriculture by increasing crop yields while drastically decreasing the application of herbicides and pesticides. This technology is necessary to allow farmers to produce sufficient food for a growing global population. Furthermore, plants are currently being engineered to produce fuel and chemical alternatives to petroleum based products. Because plants are net consumers of atmospheric carbon dioxide, they are currently being seen as a means to sequester greenhouse gases while at the same time replacing petroleum and coal as chemical feedstocks. <br />
<br><br />
<br>However, there has been recent controversy concerning the use of transgenic plants and organisms. These issues include economical, environmental, ethical, and health concerns. We have developed the following graphics outlining and discussing a short history and issues concerning GMOs. This images has been made available to help educate and for use by other participants.</p><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/d/d9/GMOs_1.png"><img src="https://static.igem.org/mediawiki/2010/d/d9/GMOs_1.png" class="shadow" style="float:center;width:400px;margin:10px"></a><br />
</html><br />
<html><a href="https://static.igem.org/mediawiki/2010/e/ed/GMOs_2.png"><img src="https://static.igem.org/mediawiki/2010/e/ed/GMOs_2.png" class="shadow" style="float:center;width:400px;margin:10px"></a><br />
</html><br />
<html><a href="https://static.igem.org/mediawiki/2010/6/61/GMOS_3.png"><img src="https://static.igem.org/mediawiki/2010/6/61/GMOS_3.png" class="shadow" style="float:center;width:400px;margin:10px"></a><br />
</html><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 />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Hilaryahttp://2010.igem.org/File:GMOS_3.pngFile:GMOS 3.png2010-10-27T19:22:02Z<p>Hilarya: </p>
<hr />
<div></div>Hilaryahttp://2010.igem.org/File:GMOs_2.pngFile:GMOs 2.png2010-10-27T19:20:52Z<p>Hilarya: </p>
<hr />
<div></div>Hilaryahttp://2010.igem.org/File:GMOs_1.pngFile:GMOs 1.png2010-10-27T19:19:49Z<p>Hilarya: </p>
<hr />
<div></div>Hilaryahttp://2010.igem.org/Team:Nevada/Transgenic_PlantsTeam:Nevada/Transgenic Plants2010-10-27T19:19:31Z<p>Hilarya: /* Transgenic Plants: into the Wild */</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 />
<p>'''Technological Advances from Genetically Engineered Plants'''<br />
<br><br />
Since the initial development of Agrobacterium transformation systems, many plant species including tobacco, tomato, potato, rice, soybean, mint, melon, cucumber, pine and poplar trees, and many others have been transformed using this ingenious bacterial vector. Important traits have been engineered into plants including pest and weed resistance, increased nutritional value, environmental stress tolerance, the production of pharmaceutical and industrial proteins, and the production of bioactive secondary chemical compounds. Our ability to genetically engineer plants has revolutionized agriculture by increasing crop yields while drastically decreasing the application of herbicides and pesticides. This technology is necessary to allow farmers to produce sufficient food for a growing global population. Furthermore, plants are currently being engineered to produce fuel and chemical alternatives to petroleum based products. Because plants are net consumers of atmospheric carbon dioxide, they are currently being seen as a means to sequester greenhouse gases while at the same time replacing petroleum and coal as chemical feedstocks. <br />
<br><br />
<br>However, there has been recent controversy concerning the use of transgenic plants and organisms. These issues include economical, environmental, ethical, and health concerns. We have developed the following graphics outlining and discussing a short history and issues concerning GMOs. This images has been made available to help educate and for use by other participants.</p><br />
<br />
<html><a href=""><img src="" class="shadow" style="float:center;width:400px;margin:10px"></a><br />
</html><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>Hilaryahttp://2010.igem.org/Team:Nevada/plant_compatible_reportersTeam:Nevada/plant compatible reporters2010-10-27T04:40:50Z<p>Hilarya: /* Reporters */</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 />
<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><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><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><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 />
{| 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>Hilaryahttp://2010.igem.org/Team:Nevada/plant_compatible_reportersTeam:Nevada/plant compatible reporters2010-10-27T04:40:25Z<p>Hilarya: /* Reporters */</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 />
<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><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><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><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 />
{| 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>Hilaryahttp://2010.igem.org/Team:Nevada/plant_compatible_reportersTeam:Nevada/plant compatible reporters2010-10-27T04:39:38Z<p>Hilarya: /* Reporters */</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 />
<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 />
'''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 />
'''Kozak, M.''' (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribososomes. ''Cell''. 44: 283-92.<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 />
{| 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>Hilaryahttp://2010.igem.org/Team:Nevada/Team_Nevada:_Plant_SummitTeam:Nevada/Team Nevada: Plant Summit2010-10-27T04:34:50Z<p>Hilarya: /* 2010 iGEM Plant Summit */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Plant Summit UNR.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<br />
== 2010 iGEM Plant Summit ==<br />
<br><br />
<p>On '''Sunday November 7th''', the iGEM Nevada Team is organizing the first '''iGEM Plant Summit'''. The goal of this summit is to develop a network of iGEM investigators working on plant systems that can help promote the adoption of plants by the larger iGEM community. We will discuss the initiation of a coordinated effort to develop iGEM compatible resources (i.e. plant promoters, reporter genes, and transformation vectors) and standardized plant transformation, maintenance and safety protocols. The pros and cons of various plant systems will be discussed. Furthermore, because, genetically engineered plants require more time to create than genetically engineered bacteria or fungi, we will also discuss strategies that will allow for the completion of plant related projects within the time constraint of the iGEM competition. <br />
</p><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>Hilarya