http://2009.igem.org/wiki/index.php?title=Special:Contributions/Bswiniar&feed=atom&limit=50&target=Bswiniar&year=&month=2009.igem.org - User contributions [en]2024-03-29T07:56:31ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Minnesota/NotebookTeam:Minnesota/Notebook2010-06-23T21:31:29Z<p>Bswiniar: Undo revision 169839 by Bswiniar (Talk)</p>
<hr />
<div>{|align="center" cellpadding="25"<br />
|[[Image:mnlogo.jpg|300px|center|frame]]<br />
|}<br />
<br />
{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"<br />
{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"<br />
!align="center"|[[Team:Minnesota|<font color="gold">Home</font>]]<br />
!align="center"|[[Team:Minnesota/Team|<font color="gold">The Team</font>]]<br />
!align="center"|[[Team:Minnesota/Project|<font color="gold">The Project</font>]]<br />
!align="center"|[[Team:Minnesota/Designer|<font color="gold">SynBioSS Designer</font>]]<br />
!align="center"|[[Team:Minnesota/Modeling|<font color="gold">Modeling</font>]]<br />
!align="center"|[[Team:Minnesota/Notebook|<font color="gold">Experimental</font>]]<br />
!align="center"|[[Team:Minnesota/Parts Characterization|<font color="gold">Competition Requirements</font>]]<br />
|}<br />
<h2>Overview</h2><br />
This year’s AND gate project is a continuation of previous years. In the past different combinations of Tet, Lac, and null operator sites were made to produce an AND gate. From last year results, we decided to use the Tet Tet Lac promoter because it was the least leaky. We decided to mutate the palindromic sequence in the Tet operator site. This would alter the binding affinity of TetR for the operator site. By doing this, we can have the modeling team more accurately predict the behavior of our promoter. We also made and tested the TNN and TTN constructs in the hopes that the models would add together thermodynamically to be equivalent to our AND gate data.<br />
<br />
<h2>Standard Protocols We Used in the Wet Lab</h2><br />
<br />
<h5>These link to pdf files:</h5><br />
<br />
[[Media:Bacterial_Culture.pdf|Bacterial Culture Protocols]]<br />
<br />
[[Media:Transformation_of_Chemically_Competent_Cells.pdf|Transformation of Chemically Competent Cells]]<br />
<br />
[[Media:Plasmid_Prep_from_Cultures.pdf|Plasmid Prep from Cultures]]<br />
<br />
[[Media:DNA_Quantification.pdf|DNA Quantification]]<br />
<br />
[[Media:PCR.pdf|Polymerase Chain Reaction (PCR)]]<br />
<br />
[[Media:Restriction_Digest.pdf|Restriction Digest]]<br />
<br />
[[Media:Vector_Dephosphorylation.pdf|Vector Dephosphorylation]]<br />
<br />
[[Media:DNA_Fragment_Ligation.pdf|DNA Fragment Ligation]]<br />
<br />
[[Media:DNA_Purification.pdf|DNA Purification]]<br />
<br />
[[Media:Sequencing.pdf|Sequencing]]<br />
<br />
[[Media:Preparing_Competent_Cells.pdf|Preparing Competent Cells]]<br />
<br />
[[Media:Soeing_PCR.pdf|SOEing PCR]]<br />
<br />
[[Media:Ligation_Reaction.pdf|Ligation]]<br />
<br />
[[Media:Screening.pdf|Screening]]<br />
<br />
[[Media:Sample_Collection.pdf|Sample Collection]]<br />
<br /><br />
<h2>Procedure</h2><br />
<br />
<ol><br />
<li>[https://static.igem.org/mediawiki/2009/4/4a/Preparing_Competent_Cells.pdf Prepare Competent Cells] for TOP10 and DH5αPro</li><br />
<li>Design the primers so that the proper mutations exist in the palindromic sequence of the Tet operator site. Design for each construct: TNN, TTN, and TTL.</li><br />
<li>Combine [https://static.igem.org/mediawiki/2009/a/ae/Soeing_PCR.pdf Soeing PCR] reagents with primers and place in a thermocycler.</li><br />
<li>Amplify the insert using [https://static.igem.org/mediawiki/2009/7/74/PCR.pdf PCR]</li><br />
<li>Purify the PCR product using [https://static.igem.org/mediawiki/2009/a/a8/DNA_Purification.pdf QIAquick PCR purification]</li><br />
<li>Ligate[https://static.igem.org/mediawiki/2009/b/b3/Ligation_Reaction.pdf Ligation] the insert into pGLOTopo</li><br />
<li>The plasmid is [https://static.igem.org/mediawiki/2009/f/f5/Transformation_of_Chemically_Competent_Cells.pdf transformed] into TOP10 cells</li><br />
<li>The transformants are plated then screened [https://static.igem.org/mediawiki/2009/6/66/Screening.pdf screened] using a florescent camera</li><br />
<li>Once the colonies have been screened, positive colonies have their plasmids isolated[https://static.igem.org/mediawiki/2009/3/39/Plasmid_Prep_from_Cultures.pdf plasmids isolated].</li><br />
<li>The plasmids are then [https://static.igem.org/mediawiki/2009/b/b0/Sequencing.pdf sequenced] to ensure the correct sequence was obtained.</li><br />
<li>Transform the cells into DH5αPro and TOP10 and perform [https://static.igem.org/mediawiki/2009/3/32/Sample_Collection.pdf sample collection]</li><br />
<li>The samples are left at 4 °C for 24 hours.</li><br />
<li>The relative GFP intensity is taken with FACSCalibar flow cytometer.</li><br />
</ol><br />
<br />
<br /><br />
<h2>Discussion of General Trends Found</h2><br />
<br />
The positive control showing a weak signal can be explained by the GFP forming inclusion bodies in high concentrations. The inclusion bodies would be fluorescently inactive and show a negative GFP signal. This theory is also support by Figure 1.C which shows two peaks for two time points. At the earlier time point, the negative GFP peak is weak while the positive gives a strong signal, however at the later time point the negative GFP signal is stronger and the positive GFP. There is also roughly the same area under both peaks meaning that it is likely that the counts from one event are being transferred to the other. Since time is the only factor and GFP accumulates over time, it can be inferred that at high concentration the cell forms the fluorescently inactive inclusion bodies. The large increase from an aTc concentration of 10 ng to 50 ng that is observed is likely explained by the saturation of TetR. With the TetR bound to its inducers, it wouldn’t bind to the operator site and produce a greater GFP intensity. However, the rate of GFP production in the TTL construct seems to be governed more strongly by the Lac operator site. This is likely due to LacR being a very large molecule which prevents polymerase from binding to the operator site.<br />
The mutations in the Tet operator site were made to change the binding affinity of TetR for the operator site. These alterations not only affect the binding affinity of just the repressor protein but also the inducer-repressor protein complex. This means that not only the leakiness of the promoter will be affected. The efficiency of repression is also affected. Our results from the AND gate expression did not support what was found when just studying just mutations in the Tet operator . The highest expression was observed with mutant 4 while mutant 3 and 5 had a slight decrease in efficiency of transcription.<br />
<br />
<br />
<br />
[[Image:IGem_4.jpg|350px|left|TTL T0P10]][[Image:IGem_2.jpg|350px|right]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:IGem_1.jpg|350px|center]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<h2>Safety</h2><br />
1. Would any of your project ideas raise safety issues in terms of: <br />
researcher safety, public safety, or environmental safety?<br />
<br /><br />
No, our constructs are simple promoter regulatory elements, built from well characterized lactose and tetracycline operon components, and characterized in non-pathogenic strain of E. coli. <br />
<br /><br />
2. Is there a local biosafety group, committee, or review board at your institution? <br />
<br /><br />
Yes, there is an Institutional Biosafety Committee at the University of Minnesota. We have described our work and gotten permission to work in a wet lab (IBC Code Number: 0706H11321).<br />
<br /><br />
3. What does your local biosafety group think about your project? <br />
<br /><br />
We use standard molecular biology techniques. IBC readily approved a continuing review.<br />
<br /><br />
4. Do any of the new BioBrick parts that you made this year raise any safety issues? If yes, did you document these issues in the Registry? <br />
<br /><br />
No, none of the parts we built raise any safety issues.<br />
<br />
<br /><br />
<html><br />
<body><br />
<br />
<h2>Notebook</h2><br />
This calendar contains a day-by-day catalog of what we did in the wet lab for our project and parts characterization. The calendar for computational work can be found below and on the <br />
<a href="https://2009.igem.org/Team:Minnesota/Modeling">Modeling</a> page.<br />
Please click on each event to see a detailed description of what we did. <br />
<br /><br />
<br />
<br /><br />
<br /><br />
</body><br />
</html><br />
<br />
<html><br />
<style type="text/css"><br />
table.calendar { margin: 0; padding: 2px; }<br />
table.calendar td { margin: 0; padding: 1px; vertical-align: top; }<br />
table.month .heading td { padding:1px; background-color:#FFFFFF; color:maroon; text-align:center; font-size:120%; font-weight:bold; }<br />
table.month .dow td { color:maroon; text-align:center; font-size:110%; }<br />
table.month td.today { background-color:#AAAAAA; }<br />
table.month td {<br />
border: none;<br />
margin: 0;<br />
padding: 0pt 1.5pt;<br />
font-weight: bold;<br />
font-size: 8pt;<br />
text-align: right;<br />
background-color: #FFFFFF;<br />
}<br />
#bodyContent table.month a { background:none; padding:0 }<br />
.day-active { color:red }<br />
.day-empty { color:black }<br />
<br />
</style> <br />
</html><br />
<br />
<br />
{| align="center"<br />
{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="10" bordercolor="#00CCFF" width="62%" align="center"<br />
!align="center"|{{#calendar: title=Minnesota-experimental |year=2009 | month=06}}<br />
!align="center"|{{#calendar: title=Minnesota-experimental |year=2009 | month=07}}<br />
!align="center"|{{#calendar: title=Minnesota-experimental |year=2009 | month=08}}<br />
!align="center"|{{#calendar: title=Minnesota-experimental |year=2009 | month=09}}<br />
!align="center"|{{#calendar: title=Minnesota-experimental |year=2009 | month=10}}<br />
|}</div>Bswiniarhttp://2009.igem.org/Team:Minnesota/NotebookTeam:Minnesota/Notebook2010-06-23T21:30:54Z<p>Bswiniar: </p>
<hr />
<div>{|align="center" cellpadding="25"<br />
|[[Image:mnlogo.jpg|300px|center|frame]]<br />
|}<br />
<br />
{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"<br />
{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"<br />
!align="center"|[[Team:Minnesota|<font color="gold">Home</font>]]<br />
!align="center"|[[Team:Minnesota/Team|<font color="gold">The Team</font>]]<br />
!align="center"|[[Team:Minnesota/Project|<font color="gold">The Project</font>]]<br />
!align="center"|[[Team:Minnesota/Designer|<font color="gold">SynBioSS Designer</font>]]<br />
!align="center"|[[Team:Minnesota/Modeling|<font color="gold">Modeling</font>]]<br />
!align="center"|[[Team:Minnesota/Notebook|<font color="gold">Experimental</font>]]<br />
!align="center"|[[Team:Minnesota/Parts Characterization|<font color="gold">Competition Requirements</font>]]<br />
|}<br />
<h2>Overview</h2><br />
This year’s AND gate project is a continuation of previous years. In the past different combinations of Tet, Lac, and null operator sites were made to produce an AND gate. From last year results, we decided to use the Tet Tet Lac promoter because it was the least leaky. We decided to mutate the palindromic sequence in the Tet operator site. This would alter the binding affinity of TetR for the operator site. By doing this, we can have the modeling team more accurately predict the behavior of our promoter. We also made and tested the TNN and TTN constructs in the hopes that the models would add together thermodynamically to be equivalent to our AND gate data.<br />
<br />
<h2>Standard Protocols We Used in the Wet Lab</h2><br />
<br />
<h5>These link to pdf files:</h5><br />
<br />
[[Media:Bacterial_Culture.pdf|Bacterial Culture Protocols]]<br />
<br />
[[Media:Transformation_of_Chemically_Competent_Cells.pdf|Transformation of Chemically Competent Cells]]<br />
<br />
[[Media:Plasmid_Prep_from_Cultures.pdf|Plasmid Prep from Cultures]]<br />
<br />
[[Media:DNA_Quantification.pdf|DNA Quantification]]<br />
<br />
[[Media:PCR.pdf|Polymerase Chain Reaction (PCR)]]<br />
<br />
[[Media:Restriction_Digest.pdf|Restriction Digest]]<br />
<br />
[[Media:Vector_Dephosphorylation.pdf|Vector Dephosphorylation]]<br />
<br />
[[Media:DNA_Fragment_Ligation.pdf|DNA Fragment Ligation]]<br />
<br />
[[Media:DNA_Purification.pdf|DNA Purification]]<br />
<br />
[[Media:Sequencing.pdf|Sequencing]]<br />
<br />
[[Media:Preparing_Competent_Cells.pdf|Preparing Competent Cells]]<br />
<br />
[[Media:Soeing_PCR.pdf|SOEing PCR]]<br />
<br />
[[Media:Ligation_Reaction.pdf|Ligation]]<br />
<br />
[[Media:Screening.pdf|Screening]]<br />
<br />
[[Media:Sample_Collection.pdf|Sample Collection]]<br />
<br /><br />
<h2>Procedure</h2><br />
<br />
<ol><br />
<li>[https://static.igem.org/mediawiki/2009/4/4a/Preparing_Competent_Cells.pdf Prepare Competent Cells] for TOP10 and DH5αPro</li><br />
<li>Design the primers so that the proper mutations exist in the palindromic sequence of the Tet operator site. Design for each construct: TNN, TTN, and TTL.</li><br />
<li>Combine [https://static.igem.org/mediawiki/2009/a/ae/Soeing_PCR.pdf Soeing PCR] reagents with primers and place in a thermocycler.</li><br />
<li>Amplify the insert using [https://static.igem.org/mediawiki/2009/7/74/PCR.pdf PCR]</li><br />
<li>Purify the PCR product using [https://static.igem.org/mediawiki/2009/a/a8/DNA_Purification.pdf QIAquick PCR purification]</li><br />
<li>Ligate[https://static.igem.org/mediawiki/2009/b/b3/Ligation_Reaction.pdf Ligation] the insert into pGLOTopo</li><br />
<li>The plasmid is [https://static.igem.org/mediawiki/2009/f/f5/Transformation_of_Chemically_Competent_Cells.pdf transformed] into TOP10 cells</li><br />
<li>The transformants are plated then screened [https://static.igem.org/mediawiki/2009/6/66/Screening.pdf screened] using a florescent camera</li><br />
<li>Once the colonies have been screened, positive colonies have their plasmids isolated[https://static.igem.org/mediawiki/2009/3/39/Plasmid_Prep_from_Cultures.pdf plasmids isolated].</li><br />
<li>The plasmids are then [https://static.igem.org/mediawiki/2009/b/b0/Sequencing.pdf sequenced] to ensure the correct sequence was obtained.</li><br />
<li>Transform the cells into DH5αPro and TOP10 and perform [https://static.igem.org/mediawiki/2009/3/32/Sample_Collection.pdf sample collection]</li><br />
<li>The samples are left at 4 °C for 24 hours.</li><br />
<li>The relative GFP intensity is taken with FACSCalibar flow cytometer.</li><br />
</ol><br />
<br />
<br /><br />
<h2>Discussion of General Trends Found</h2><br />
<br />
The positive control showing a weak signal can be explained by the GFP forming inclusion bodies in high concentrations. The inclusion bodies would be fluorescently inactive and show a negative GFP signal. This theory is also support by Figure 1.C which shows two peaks for two time points. At the earlier time point, the negative GFP peak is weak while the positive gives a strong signal, however at the later time point the negative GFP signal is stronger and the positive GFP. There is also roughly the same area under both peaks meaning that it is likely that the counts from one event are being transferred to the other. Since time is the only factor and GFP accumulates over time, it can be inferred that at high concentration the cell forms the fluorescently inactive inclusion bodies. The large increase from an aTc concentration of 10 ng to 50 ng that is observed is likely explained by the saturation of TetR. With the TetR bound to its inducers, it wouldn’t bind to the operator site and produce a greater GFP intensity. However, the rate of GFP production in the TTL construct seems to be governed more strongly by the Lac operator site. This is likely due to LacR being a very large molecule which prevents polymerase from binding to the operator site.<br />
The mutations in the Tet operator site were made to change the binding affinity of TetR for the operator site. These alterations not only affect the binding affinity of just the repressor protein but also the inducer-repressor protein complex. This means that not only the leakiness of the promoter will be affected. The efficiency of repression is also affected. Our results from the AND gate expression did not support what was found when just studying just mutations in the Tet operator . The highest expression was observed with mutant 4 while mutant 3 and 5 had a slight decrease in efficiency of transcription.<br />
<br />
<br />
<br />
[[Image:IGem_4.jpg|350px|left|TTL T0P10]][[Image:IGem_2.jpg|350px|right]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:IGem_1.jpg|350px|center]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<h2>Safety</h2><br />
1. Would any of your project ideas raise safety issues in terms of: <br />
researcher safety, public safety, or environmental safety?<br />
<br /><br />
No, our constructs are simple promoter regulatory elements, built from well characterized lactose and tetracycline operon components, and characterized in non-pathogenic strain of E. coli. <br />
<br /><br />
2. Is there a local biosafety group, committee, or review board at your institution? <br />
<br /><br />
Yes, there is an Institutional Biosafety Committee at the University of Minnesota. We have described our work and gotten permission to work in a wet lab (IBC Code Number: 0706H11321).<br />
<br /><br />
3. What does your local biosafety group think about your project? <br />
<br /><br />
We use standard molecular biology techniques. IBC readily approved a continuing review.<br />
<br /><br />
4. Do any of the new BioBrick parts that you made this year raise any safety issues? If yes, did you document these issues in the Registry? <br />
<br /><br />
No, none of the parts we built raise any safety issues.<br />
<br />
<br /><br />
<html><br />
<body><br />
<br />
<h2>Notebook</h2><br />
This calendar contains a day-by-day catalog of what we did in the wet lab for our project and parts characterization. The calendar for computational work can be found below and on the <br />
<a href="https://2009.igem.org/Team:Minnesota/Modeling">Modeling</a> page.<br />
Please click on each event to see a detailed description of what we did. <br />
<br /><br />
<br />
<br /><br />
<br /><br />
</body><br />
</html><br />
<br />
<html><br />
<style type="text/css"><br />
table.calendar { margin: 0; padding: 2px; }<br />
table.calendar td { margin: 0; padding: 1px; vertical-align: top; }<br />
table.month .heading td { padding:1px; background-color:#FFFFFF; color:maroon; text-align:center; font-size:120%; font-weight:bold; }<br />
table.month .dow td { color:maroon; text-align:center; font-size:110%; }<br />
table.month td.today { background-color:#AAAAAA; }<br />
table.month td {<br />
border: none;<br />
margin: 0;<br />
padding: 0pt 1.5pt;<br />
font-weight: bold;<br />
font-size: 8pt;<br />
text-align: right;<br />
background-color: #FFFFFF;<br />
}<br />
#bodyContent table.month a { background:none; padding:0 }<br />
.day-active { color:red }<br />
.day-empty { color:black }<br />
<br />
</style> <br />
</html><br />
<br />
<br />
{| align="center"<br />
{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="10" bordercolor="#00CCFF" width="62%" align="center"<br />
!align="center"|{{#calendar: title=Minnesota-experimental |year=2010 | month=06}}<br />
!align="center"|{{#calendar: title=Minnesota-experimental |year=2010 | month=07}}<br />
!align="center"|{{#calendar: title=Minnesota-experimental |year=2010 | month=08}}<br />
!align="center"|{{#calendar: title=Minnesota-experimental |year=2010 | month=09}}<br />
!align="center"|{{#calendar: title=Minnesota-experimental |year=2010 | month=10}}<br />
|}</div>Bswiniarhttp://2009.igem.org/Team:MinnesotaTeam:Minnesota2010-06-23T20:18:26Z<p>Bswiniar: Undo revision 169835 by Bswiniar (Talk)</p>
<hr />
<div>{|align="center" cellpadding="25"<br />
|[[Image:mnlogo.jpg|300px|center|frame]]<br />
|}<br />
<br />
{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"<br />
!align="center"|[[Team:Minnesota|<font color="gold">Home</font>]]<br />
!align="center"|[[Team:Minnesota/Team|<font color="gold">The Team</font>]]<br />
!align="center"|[[Team:Minnesota/Project|<font color="gold">The Project</font>]]<br />
!align="center"|[[Team:Minnesota/Designer|<font color="gold">SynBioSS Designer</font>]]<br />
!align="center"|[[Team:Minnesota/Modeling|<font color="gold">Modeling</font>]]<br />
!align="center"|[[Team:Minnesota/Notebook|<font color="gold">Experimental</font>]]<br />
!align="center"|[[Team:Minnesota/Parts Characterization|<font color="gold">Competition Requirements</font>]]<br />
|}<br />
<br />
<br />
[[Image:teamphoto2.jpeg|400px|right]]<br />
<br />
<h1>Welcome!</h1><br />
Welcome to the Team Minnesota Wiki for iGEM 2009! <br />
<br />
We are a team of undergraduate and graduate students along with many advisors. This is the second year that Minnesota has sent a team to iGEM. Last year we were runners-up for the Best New BioBrick part (Natural) and we hope to accomplish even more with our design of a logical AND gate in <i>E. coli</i> this year. <br />
<br />
<h2>The Mission</h2><br />
<p>We are working on a very well studied AND gate, which is composed of elements of the Tet, Lac, and lambda-phage promoters and is responsive to the commonly-used inducers IPTG and aTc, producing GFP as an output signal. <br />
Well, we have decided not to ride the wave of ever-more complex synthetic constructs. Instead, we will isolate elements of the AND gate and study them individually. This will allow for a greater understanding of how exactly this AND gate works, and will eventually allow for creation of better AND gates, and a more predictable response to inputs by the biological systems.</p><br />
<p>Our group will be doing both experimental and modeling work this year. These two approachs will complement each other. The experimental data will be used to refine the mathematical models, while the models will determine good directions for the experiments to pursue. We will go into greater detail on both these aspects on theeir respective wiki pages. The approach we are taking can be applied to many other biological circuits and can lead to greater understanding of how to engineer biological circuits.</p><br />
<p> What is unique in our work is the effort to develop software tools that streamline the construction of synthetic biological systems. The Synthetic Biology Software Suite is the product of our work, and this year iGEM students are working on SynBioSS Designer, a tool that uses Registry parts to build a model of biomolecular interaction. This model can be used to simulate the dynamic behavior of the gene network.</p><br />
<br />
<h2>Highlights for the Judges</h2><br />
Judges! Please navigate to our [https://2009.igem.org/Team:Minnesota/Criteria Judging Criteria] page for a comprehensive list of UMN's fulfilled medal requirements and links throughout the wiki.</div>Bswiniarhttp://2009.igem.org/Team:MinnesotaTeam:Minnesota2010-06-23T20:17:53Z<p>Bswiniar: Undo revision 169836 by Bswiniar (Talk)</p>
<hr />
<div>{|align="center" cellpadding="25"<br />
|[[Image:mnlogo.jpg|300px|center|frame]]<br />
|}<br />
<br />
{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"<br />
!align="center"|[[Team:Minnesota|<font color="gold">Home</font>]]<br />
!align="center"|[[Team:Minnesota/Team|<font color="gold">The Team</font>]]<br />
!align="center"|[[Team:Minnesota/Project|<font color="gold">The Project</font>]]<br />
!align="center"|[[Team:Minnesota/Designer|<font color="gold">SynBioSS Designer</font>]]<br />
!align="center"|[[Team:Minnesota/Modeling|<font color="gold">Modeling</font>]]<br />
!align="center"|[[Team:Minnesota/Notebook|<font color="gold">Experimental</font>]]<br />
!align="center"|[[Team:Minnesota/Parts Characterization|<font color="gold">Competition Requirements</font>]]<br />
|}<br />
<br />
<br />
[[Image:teamphoto2.jpeg|400px|right]]<br />
<br />
<h1>Welcome!</h1><br />
Welcome to the Team Minnesota Wiki for iGEM 2010! <br />
<br />
We are a team of undergraduate and graduate students along with many advisors. This is the third year that Minnesota has sent a team to iGEM. Check back later for more updates on our project!<br />
<br />
<h2>The Mission</h2><br />
<p>We are working on a very well studied AND gate, which is composed of elements of the Tet, Lac, and lambda-phage promoters and is responsive to the commonly-used inducers IPTG and aTc, producing GFP as an output signal. <br />
Well, we have decided not to ride the wave of ever-more complex synthetic constructs. Instead, we will isolate elements of the AND gate and study them individually. This will allow for a greater understanding of how exactly this AND gate works, and will eventually allow for creation of better AND gates, and a more predictable response to inputs by the biological systems.</p><br />
<p>Our group will be doing both experimental and modeling work this year. These two approachs will complement each other. The experimental data will be used to refine the mathematical models, while the models will determine good directions for the experiments to pursue. We will go into greater detail on both these aspects on theeir respective wiki pages. The approach we are taking can be applied to many other biological circuits and can lead to greater understanding of how to engineer biological circuits.</p><br />
<p> What is unique in our work is the effort to develop software tools that streamline the construction of synthetic biological systems. The Synthetic Biology Software Suite is the product of our work, and this year iGEM students are working on SynBioSS Designer, a tool that uses Registry parts to build a model of biomolecular interaction. This model can be used to simulate the dynamic behavior of the gene network.</p><br />
<br />
<h2>Highlights for the Judges</h2><br />
Judges! Please navigate to our [https://2009.igem.org/Team:Minnesota/Criteria Judging Criteria] page for a comprehensive list of UMN's fulfilled medal requirements and links throughout the wiki.</div>Bswiniarhttp://2009.igem.org/Team:MinnesotaTeam:Minnesota2010-06-23T20:10:32Z<p>Bswiniar: </p>
<hr />
<div>{|align="center" cellpadding="25"<br />
|[[Image:mnlogo.jpg|300px|center|frame]]<br />
|}<br />
<br />
{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"<br />
!align="center"|[[Team:Minnesota|<font color="gold">Home</font>]]<br />
!align="center"|[[Team:Minnesota/Team|<font color="gold">The Team</font>]]<br />
!align="center"|[[Team:Minnesota/Project|<font color="gold">The Project</font>]]<br />
!align="center"|[[Team:Minnesota/Designer|<font color="gold">SynBioSS Designer</font>]]<br />
!align="center"|[[Team:Minnesota/Modeling|<font color="gold">Modeling</font>]]<br />
!align="center"|[[Team:Minnesota/Notebook|<font color="gold">Experimental</font>]]<br />
!align="center"|[[Team:Minnesota/Parts Characterization|<font color="gold">Competition Requirements</font>]]<br />
|}<br />
<br />
<br />
[[Image:teamphoto2.jpeg|400px|right]]<br />
<br />
<h1>Welcome!</h1><br />
Welcome to the Team Minnesota Wiki for iGEM 2010! <br />
<br />
We are a team of undergraduate and graduate students along with many advisors. This is the third year that Minnesota has sent a team to iGEM. Check back later for more updates on our project!<br />
<br />
<h2>The Mission</h2><br />
<br />
<h2>Highlights for the Judges</h2><br />
Judges! Please navigate to our [https://2010.igem.org/Team:Minnesota/Criteria Judging Criteria] page for a comprehensive list of UMN's fulfilled medal requirements and links throughout the wiki.</div>Bswiniarhttp://2009.igem.org/Team:MinnesotaTeam:Minnesota2010-06-23T20:08:00Z<p>Bswiniar: </p>
<hr />
<div>{|align="center" cellpadding="25"<br />
|[[Image:mnlogo.jpg|300px|center|frame]]<br />
|}<br />
<br />
{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"<br />
!align="center"|[[Team:Minnesota|<font color="gold">Home</font>]]<br />
!align="center"|[[Team:Minnesota/Team|<font color="gold">The Team</font>]]<br />
!align="center"|[[Team:Minnesota/Project|<font color="gold">The Project</font>]]<br />
!align="center"|[[Team:Minnesota/Designer|<font color="gold">SynBioSS Designer</font>]]<br />
!align="center"|[[Team:Minnesota/Modeling|<font color="gold">Modeling</font>]]<br />
!align="center"|[[Team:Minnesota/Notebook|<font color="gold">Experimental</font>]]<br />
!align="center"|[[Team:Minnesota/Parts Characterization|<font color="gold">Competition Requirements</font>]]<br />
|}<br />
<br />
<br />
[[Image:teamphoto2.jpeg|400px|right]]<br />
<br />
<h1>Welcome!</h1><br />
Welcome to the Team Minnesota Wiki for iGEM 2010! <br />
<br />
We are a team of undergraduate and graduate students along with many advisors. This is the third year that Minnesota has sent a team to iGEM. Check back later for more updates on our project!<br />
<br />
<h2>The Mission</h2><br />
<p>We are working on a very well studied AND gate, which is composed of elements of the Tet, Lac, and lambda-phage promoters and is responsive to the commonly-used inducers IPTG and aTc, producing GFP as an output signal. <br />
Well, we have decided not to ride the wave of ever-more complex synthetic constructs. Instead, we will isolate elements of the AND gate and study them individually. This will allow for a greater understanding of how exactly this AND gate works, and will eventually allow for creation of better AND gates, and a more predictable response to inputs by the biological systems.</p><br />
<p>Our group will be doing both experimental and modeling work this year. These two approachs will complement each other. The experimental data will be used to refine the mathematical models, while the models will determine good directions for the experiments to pursue. We will go into greater detail on both these aspects on theeir respective wiki pages. The approach we are taking can be applied to many other biological circuits and can lead to greater understanding of how to engineer biological circuits.</p><br />
<p> What is unique in our work is the effort to develop software tools that streamline the construction of synthetic biological systems. The Synthetic Biology Software Suite is the product of our work, and this year iGEM students are working on SynBioSS Designer, a tool that uses Registry parts to build a model of biomolecular interaction. This model can be used to simulate the dynamic behavior of the gene network.</p><br />
<br />
<h2>Highlights for the Judges</h2><br />
Judges! Please navigate to our [https://2009.igem.org/Team:Minnesota/Criteria Judging Criteria] page for a comprehensive list of UMN's fulfilled medal requirements and links throughout the wiki.</div>Bswiniarhttp://2009.igem.org/Minnesota/2_July_2009Minnesota/2 July 20092009-08-25T16:48:09Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/1 July 2009|Go to Previous Day (July 1)]]'''|| width=158|'''[[Minnesota/3 July 2009|Go to Next Day (July 3)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I re-looked at some of the kinetic factors that we can change to see if changing the model may have affected how they effect the graph. I submitted eight models today as described below:<br><br />
<br />
'''smad_ttn_tetRo_u/d''' - These two models looked at the affects of changing the rate of tetR2 being eliminated from the cell. Specifically reactions 35, 36, 37 mentioned before. The kinetic constants were moved up or down by an order of magnitude (k = 0.00028881 1/s initially).<br><br />
<br />
'''smad_ttn_tetRin_u/d''' - These two models looked at the affects of changing the rate of synthesis of tetR2 (reaction 44) in the cell, by either moving the kinetic constant up an order of magnitude (1E-11 to 1E-10), or down an order of magnitude (1E-11 to 1E-12).<br><br />
<br />
'''smad_ttn_aff_u/d''' - These two models changed the kinetic constants for tetR2 affinity for tetO1 and tetO2. The reactions affected are reaction 16, 18, 20, 26, 28, 30 as mentioned before. Moving the kinetic constants (which range from 0.001 1/s to 10000 1/s) up will allow tetO1 and tetO2 to release from tetR2 more easily. Moving the kinetic constants down will make tetO1 and tetO2 bind more tightly.<br><br />
<br />
'''smad_ttn_test1/2''' - These two models extend the smad_ttn_tetRin_*.nc models. For Test2 I looked at changing reactions 35, 36, 37, and also 42 and 43 down an order of magnitude. These reactions represent every way tetR2 can be permanently eliminated from the cell (besides dilution from cell division). Test1 also moved reaction 44 (--> TetR2, synthesis of tetR2) down an order of magnitude.<br><br />
<br />
The results for these models are expected tomorrow.<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky17.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
The major problem in the model has been solved! The model no longer shows a hump and a decrease by 12 hours. Instead, the model reaches a plateau at 6 hours or so, which is what was expected. This was due to an error in the species that split on division of the cell. Instead of GFP splitting on cell division, a different species was, which caused the GFP to decrease over time. This change also causes the model to oscillate as well, which is due to the cells not splitting all at the same time. This is of little concern to the accuracy of the model, although it is probably no exactly what happens in real cells.</div>Bswiniarhttp://2009.igem.org/Minnesota/2_July_2009Minnesota/2 July 20092009-08-25T16:36:13Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/1 July 2009|Go to Previous Day (July 1)]]'''|| width=158|'''[[Minnesota/3 July 2009|Go to Next Day (July 3)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I re-looked at some of the kinetic factors that we can change to see if changing the model may have affected how they effect the graph. I submitted eight models today as described below:<br><br />
<br />
'''smad_ttn_tetRo_u/d''' - These two models looked at the affects of changing the rate of tetR2 being eliminated from the cell. Specifically reactions 35, 36, 37 mentioned before. The kinetic constants were moved up or down by an order of magnitude (k = 0.00028881 1/s initially).<br><br />
<br />
'''smad_ttn_tetRin_u/d''' - These two models looked at the affects of changing the rate of synthesis of tetR2 (reaction 44) in the cell, by either moving the kinetic constant up an order of magnitude (1E-11 to 1E-10), or down an order of magnitude (1E-11 to 1E-12).<br><br />
<br />
'''smad_ttn_aff_u/d''' - These two models changed the kinetic constants for tetR2 affinity for tetO1 and tetO2. The reactions affected are reaction 16, 18, 20, 26, 28, 30 as mentioned before. Moving the kinetic constants (which range from 0.001 1/s to 10000 1/s) up will allow tetO1 and tetO2 to release from tetR2 more easily. Moving the kinetic constants down will make tetO1 and tetO2 bind more tightly.<br><br />
<br />
'''smad_ttn_test1/2''' - These two models extend the smad_ttn_tetRin_*.nc models. For Test2 I looked at changing reactions 35, 36, 37, and also 42 and 43 down an order of magnitude. These reactions represent every way tetR2 can be permanently eliminated from the cell (besides dilution from cell division). Test1 also moved reaction 44 (--> TetR2, synthesis of tetR2) down an order of magnitude.<br><br />
<br />
The results for these models are expected tomorrow.<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky17.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
The major problem in the model has been solved! The model no longer shows a hump and a decrease by 12 hours. Instead, the model reaches a plateau at 6 hours or so, which is what was expected. This was due to an error in the species that split on division of the cell.</div>Bswiniarhttp://2009.igem.org/Minnesota/1_July_2009Minnesota/1 July 20092009-08-25T16:27:37Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/30 June 2009|Go to Previous Day (June 30)]]'''|| width=158|'''[[Minnesota/2 July 2009|Go to Next Day (July 2)]]'''<br />
|}<br />
'''Patrick'''<br><br />
A major issue was uncovered with the model being used. Specifically, designGUI, when deleting reactions, will delete unused species, which is good. The GUI though also does not update which species number is being "Split on Division" (SOD). This resulted in RNAp being split every time the cells divided. Since it wasn't being produced, the number of RNAp per cell would continue to decrease as time went on, which would result in lower GFP production than expected.<br><br />
<br />
Today I ran the base model after checking to make sure, finally, that everything was as it was expected to be, including the species being split on division:<br><br />
<center><gallery widths=400 heights=300><br />
Image:Base.jpg|Figure 1 - Base TTN Model<br />
</gallery></center><br><br />
<br />
'''Ben'''<br><br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||623000||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||623000||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||623000||<br />
|}<br />
[[Image:Tnnatcvariesleaky15.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
In this model, the constant was increased drastically to 6.23E+05, which definitely decreases the leakiness of the model. However, the model still shows a hump, which is definitely incorrect.</div>Bswiniarhttp://2009.igem.org/Minnesota/30_June_2009Minnesota/30 June 20092009-08-25T16:21:33Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/29 June 2009|Go to Previous Day (June 29)]]'''|| width=158|'''[[Minnesota/1 July 2009|Go to Next Day (July 1)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Here is the "leaky" graph along with the base for comparison:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:badBase.jpg|Figure 1 - Base TTN Model<br />
Image:Leak.jpg|Figure 2 - Leaky Model<br />
</gallery></center><br><br />
<br />
As can be seen the "leaky" graph indeed does show increased GFP production at low aTc concentrations.<br><br />
<br />
'''Ben'''<br><br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||30||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||30||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||30||<br />
|}<br />
[[Image:Tnnatcvariesleaky11.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
These graphs show almost no change from yesterday's graph, even though the kinetic constant has been reduced by an order of magnitude. This is probably due to the fact that the constant is so low already, and so making it even lower will have little effect.</div>Bswiniarhttp://2009.igem.org/Minnesota/29_June_2009Minnesota/29 June 20092009-08-25T16:19:28Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/26 June 2009|Go to Previous Day (June 26)]]'''|| width=158|'''[[Minnesota/30 June 2009|Go to Next Day (June 30)]]'''<br />
|}<br />
'''Patrick'''<br><br />
I switched over today to looking at making the "leakiness" equations for the TTN model. Initially I would represent the leakiness with two equations:<br><br />
<br />
<center><br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Rxn #<br />
! Reaction<br />
! Forward Kinetic Constant<br />
|-<br />
|46||RNAp + lacP + tetO1:tetR2 + tetO2 -> RNAp:lacP||1E+07<br />
|-<br />
|47||RNAp + lacP + tetO1 + tetO2:tetR2 -> RNAp:lacP||1E+07<br />
|}</center><br><br />
<br />
The aTc complexes don't have to be represented since the leakiness is for aTc=0 in particular. Also a third leakiness equation could be made, one where both tetO1:tetR2 and tetO2:tetR2 exist, but I chose to add this later if necessary. I expect the results tomorrow.<br><br />
<br />
'''Ben'''<br><br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||310||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||310||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||310||<br />
|}<br />
[[Image:Tnnatcvariesleaky9.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
The table above shows the changes that were made from the base model inorder to produce the modeling graph shown above. This model was completed by the supercomputer the day after the previous model, by which time I knew this direction was not giving good results. The low kinetic constant for these three reactions simply produces low amounts of GFP in the system at all times except for when the system has no aTc in it.</div>Bswiniarhttp://2009.igem.org/Minnesota/29_June_2009Minnesota/29 June 20092009-08-25T16:16:50Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/26 June 2009|Go to Previous Day (June 26)]]'''|| width=158|'''[[Minnesota/30 June 2009|Go to Next Day (June 30)]]'''<br />
|}<br />
'''Patrick'''<br><br />
I switched over today to looking at making the "leakiness" equations for the TTN model. Initially I would represent the leakiness with two equations:<br><br />
<br />
<center><br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Rxn #<br />
! Reaction<br />
! Forward Kinetic Constant<br />
|-<br />
|46||RNAp + lacP + tetO1:tetR2 + tetO2 -> RNAp:lacP||1E+07<br />
|-<br />
|47||RNAp + lacP + tetO1 + tetO2:tetR2 -> RNAp:lacP||1E+07<br />
|}</center><br><br />
<br />
The aTc complexes don't have to be represented since the leakiness is for aTc=0 in particular. Also a third leakiness equation could be made, one where both tetO1:tetR2 and tetO2:tetR2 exist, but I chose to add this later if necessary. I expect the results tomorrow.<br><br />
<br />
'''Ben'''<br><br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||310||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||310||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||310||<br />
|}<br />
[[Image:Tnnatcvariesleaky9.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/Minnesota/26_June_2009Minnesota/26 June 20092009-07-30T21:23:54Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/25 June 2009|Go to Previous Day (June 25)]]'''|| width=158|'''[[Minnesota/29 June 2009|Go to Next Day (June 29)]]'''<br />
|}<br />
This model (tnn_atcvaries7_leaky) was an attempt to see what would happen if the kinetic constant was decreased for the three reactions shown below, instead of increased like the previous day's. <br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||31000||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||31000||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||31000||<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky7.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
The result of this change was quite interesting. The curve at aTc = 0 remained constant, while the entire rest of the model has been lowered. This precisely the opposite of what is needed for the model unfortunately. This direction will probably not be pursued in the future as it is not helping solve the porblems seen in the model.</div>Bswiniarhttp://2009.igem.org/Minnesota/26_June_2009Minnesota/26 June 20092009-07-30T21:13:13Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/25 June 2009|Go to Previous Day (June 25)]]'''|| width=158|'''[[Minnesota/29 June 2009|Go to Next Day (June 29)]]'''<br />
|}<br />
This model (tnn_atcvaries7_leaky) was an attempt to see what would happen if the kinetic constant was decreased for the three reactions shown below, instead of increased like the previous day's. <br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||31000||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||31000||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||31000||<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky7.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
The result was fairly predictable. Instead of making the model less leaky like the previous days, it increased the leakiness shown by the model. This direction is making the model worse, and so it will not be pursued in the future.</div>Bswiniarhttp://2009.igem.org/Minnesota/25_June_2009Minnesota/25 June 20092009-07-30T21:03:44Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/24 June 2009|Go to Previous Day (June 24)]]'''|| width=158|'''[[Minnesota/26 June 2009|Go to Next Day (June 26)]]'''<br />
|}<br />
In order to try to improve the model, the following reaction constants were changed to try to see less leakiness in the model.<br />
<br><br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||1200000||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||1200000||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||1200000||<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky5.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
This model does indeed show some different leakiness, which is definitely a good thing, although the leakiness is still too high for an accurate model. Still, the problem remains where the model peaks and decreases instead of reaching a steady state.</div>Bswiniarhttp://2009.igem.org/Minnesota/25_June_2009Minnesota/25 June 20092009-07-30T21:02:04Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/24 June 2009|Go to Previous Day (June 24)]]'''|| width=158|'''[[Minnesota/26 June 2009|Go to Next Day (June 26)]]'''<br />
|}<br />
In order to try to improve the model, the following reaction constants were changed to try to see less leakiness in the model.<br />
<br><br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||1200000||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||1200000||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||1200000||<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky5.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/Minnesota/25_June_2009Minnesota/25 June 20092009-07-30T21:01:44Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/24 June 2009|Go to Previous Day (June 24)]]'''|| width=158|'''[[Minnesota/26 June 2009|Go to Next Day (June 26)]]'''<br />
|}<br />
In order to try to improve the model, the following reaction constants were changed to try to see less leakiness in the model.<br />
<br><br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky5.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/Minnesota/24_June_2009Minnesota/24 June 20092009-07-30T20:58:05Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/23 June 2009|Go to Previous Day (June 23)]]'''|| width=158|'''[[Minnesota/25 June 2009|Go to Next Day (June 25)]]'''<br />
|}<br />
Since there are several problems with the first TNN model, we are deciding to focus on them one at a time. The first thing to work on is the leakiness problem. In order to deal with this issue, we are including a reaction that allows for the binding of the RNA polymerase to the operator site even if the site is occupied by a tetR. This will allow for some expression at an aTc concentration of 0, because at this point tetR is almost always bound to the tetO operator.<br />
<br />
Here is the model we are working on:<br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
| RNAp + lacP + lacI4:lacO1 -> RNAp:lacP||6.23E+05|| <br />
|-<br />
| RNAp + lacP + lacO1 ↔ RNAp:lacP||1E+07|| 1<br />
|-<br />
|RNA:lacP -> RNAp:lacP* || .01 ||<br />
|-<br />
|RNAp:lacP* -> lacP + lacO1 + RNAp:DNAgfp||30||<br />
|-<br />
|RNAp:DNAgfp -> RNAp + gfp_mRNA||30||<br />
|-<br />
|gfp_mRNA + rib -> rib:gfp_mRNA||100000||<br />
|-<br />
|rib:gfp_mRNA -> rib:gfp_mRNA_1 + gfp_mRNA||33||<br />
|-<br />
|rib:gfp_mRNA_1 -> rib + gfp||33||<br />
|-<br />
|tetR2 + aTc ↔ tetR2:aTc||2E+09||4E-04<br />
|-<br />
|tetR2:aTc + aTc ↔ tetR2:aTc2||1E+08||1E-03<br />
|-<br />
|tetR2:aTc + tetO1 ↔ tetR2:tetO1:aTc||1E+08||1<br />
|-<br />
|tetR2:aTc2 + tetO1 ↔ tetR2:tetO1:aTc2||1E+08||1E+05<br />
|-<br />
|tetR2:tetO1 + aTc ↔ tetR2:tetO1:aTc||1E+08||1E-03<br />
|-<br />
|tetR2:tetO1:aTc + aTc ↔ tetR2:tetO1:aTc2||1E+08||1E-03<br />
|-<br />
|tetR2 -> Ø||2.89E-04||<br />
|-<br />
|tetR2:aTc -> aTc||2.89E-04||<br />
|-<br />
|tetR2:aTc2 -> 2 aTc||2.89E-04||<br />
|-<br />
|tetR2 + nsDNA ↔ tetR2:nsDNA||1000||3.2409<br />
|-<br />
|tetR2:aTc + nsDNA ↔ tetR2:aTc:nsDNA||1000||3.2409<br />
|-<br />
|tetR2:aTc:nsDNA -> aTc + nsDNA||1.93E-04||<br />
|-<br />
|tetR2:nsDNA -> nsDNA||1.93E-04||<br />
|-<br />
|Ø -> tetR2||1E-11||<br />
|-<br />
|aTc_ext -> aTc||3.3E-04||<br />
|-<br />
|gfp_mRNA -> Ø||1.16E-03||<br />
|-<br />
|gfp -> Ø||3.21E-05||<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky3.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
As you can see from the above graph, this model shows no response to aTc concentration. The gfp output is the same whether there are 0 or 260 molecules of aTc in the cell. Essentially this system is completely leaky, which is not what is needed. Also, the model still peaks and decreases, instead of plateauing.</div>Bswiniarhttp://2009.igem.org/Minnesota/24_June_2009Minnesota/24 June 20092009-07-30T20:55:45Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/23 June 2009|Go to Previous Day (June 23)]]'''|| width=158|'''[[Minnesota/25 June 2009|Go to Next Day (June 25)]]'''<br />
|}<br />
Since there are several problems with the first TNN model, we are deciding to focus on them one at a time. The first thing to work on is the leakiness problem. In order to deal with this issue, we are including a reaction that allows for the binding of the RNA polymerase to the operator site even if the site is occupied by a tetR. This will allow for some expression at an aTc concentration of 0, because at this point tetR is almost always bound to the tetO operator.<br />
<br />
Here is the model we are working on:<br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
| RNAp + lacP + lacI4:lacO1 -> RNAp:lacP||6.23E+05|| <br />
|-<br />
| RNAp + lacP + lacO1 ↔ RNAp:lacP||1E+07|| 1<br />
|-<br />
|RNA:lacP -> RNAp:lacP* || .01 ||<br />
|-<br />
|RNAp:lacP* -> lacP + lacO1 + RNAp:DNAgfp||30||<br />
|-<br />
|RNAp:DNAgfp -> RNAp + gfp_mRNA||30||<br />
|-<br />
|gfp_mRNA + rib -> rib:gfp_mRNA||100000||<br />
|-<br />
|rib:gfp_mRNA -> rib:gfp_mRNA_1 + gfp_mRNA||33||<br />
|-<br />
|rib:gfp_mRNA_1 -> rib + gfp||33||<br />
|-<br />
|tetR2 + aTc ↔ tetR2:aTc||2E+09||4E-04<br />
|-<br />
|tetR2:aTc + aTc ↔ tetR2:aTc2||1E+08||1E-03<br />
|-<br />
|tetR2:aTc + tetO1 ↔ tetR2:tetO1:aTc||1E+08||1<br />
|-<br />
|tetR2:aTc2 + tetO1 ↔ tetR2:tetO1:aTc2||1E+08||1E+05<br />
|-<br />
|tetR2:tetO1 + aTc ↔ tetR2:tetO1:aTc||1E+08||1E-03<br />
|-<br />
|tetR2:tetO1:aTc + aTc ↔ tetR2:tetO1:aTc2||1E+08||1E-03<br />
|-<br />
|tetR2 -> Ø||2.89E-04||<br />
|-<br />
|tetR2:aTc -> aTc||2.89E-04||<br />
|-<br />
|tetR2:aTc2 -> 2 aTc||2.89E-04||<br />
|-<br />
|tetR2 + nsDNA ↔ tetR2:nsDNA||1000||3.2409<br />
|-<br />
|tetR2:aTc + nsDNA ↔ tetR2:aTc:nsDNA||1000||3.2409<br />
|-<br />
|tetR2:aTc:nsDNA -> aTc + nsDNA||1.93E-04||<br />
|-<br />
|tetR2:nsDNA -> nsDNA||1.93E-04||<br />
|-<br />
|Ø -> tetR2||1E-11||<br />
|-<br />
|aTc_ext -> aTc||3.3E-04||<br />
|-<br />
|gfp_mRNA -> Ø||1.16E-03||<br />
|-<br />
|gfp -> Ø||3.21E-05||<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky3.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/Minnesota/24_June_2009Minnesota/24 June 20092009-07-30T20:55:25Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/23 June 2009|Go to Previous Day (June 23)]]'''|| width=158|'''[[Minnesota/25 June 2009|Go to Next Day (June 25)]]'''<br />
|}<br />
Since there are several problems with the first TNN model, we are deciding to focus on them one at a time. The first thing to work on is the leakiness problem. In order to deal with this issue, we are including a reaction that allows for the binding of the RNA polymerase to the operator site even if the site is occupied by a tetR. This will allow for some expression at an aTc concentration of 0, because at this point tetR is almost always bound to the tetO operator.<br />
<br />
Here is the model we are working on:<br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
| RNAp + lacP + lacI4:lacO1 -> RNAp:lacP||6.23E+05|| <br />
|-<br />
| RNAp + lacP + lacO1 ↔ RNAp:lacP||1E+07|| 1<br />
|-<br />
|RNA:lacP -> RNAp:lacP* || .01 ||<br />
|-<br />
|RNAp:lacP* -> lacP + lacO1 + RNAp:DNAgfp||30||<br />
|-<br />
|RNAp:DNAgfp -> RNAp + gfp_mRNA||30||<br />
|-<br />
|gfp_mRNA + rib -> rib:gfp_mRNA||100000||<br />
|-<br />
|rib:gfp_mRNA -> rib:gfp_mRNA_1 + gfp_mRNA||33||<br />
|-<br />
|rib:gfp_mRNA_1 -> rib + gfp||33||<br />
|-<br />
|tetR2 + aTc ↔ tetR2:aTc||2E+09||4E-04<br />
|-<br />
|tetR2:aTc + aTc ↔ tetR2:aTc2||1E+08||1E-03<br />
|-<br />
|tetR2:aTc + tetO1 ↔ tetR2:tetO1:aTc||1E+08||1<br />
|-<br />
|tetR2:aTc2 + tetO1 ↔ tetR2:tetO1:aTc2||1E+08||1E+05<br />
|-<br />
|tetR2:tetO1 + aTc ↔ tetR2:tetO1:aTc||1E+08||1E-03<br />
|-<br />
|tetR2:tetO1:aTc + aTc ↔ tetR2:tetO1:aTc2||1E+08||1E-03<br />
|-<br />
|tetR2 -> Ø||2.89E-04||<br />
|-<br />
|tetR2:aTc -> aTc||2.89E-04||<br />
|-<br />
|tetR2:aTc2 -> 2 aTc||2.89E-04||<br />
|-<br />
|tetR2 + nsDNA ↔ tetR2:nsDNA||1000||3.2409<br />
|-<br />
|tetR2:aTc + nsDNA ↔ tetR2:aTc:nsDNA||1000||3.2409<br />
|-<br />
|tetR2:aTc:nsDNA -> aTc + nsDNA||1.93E-04||<br />
|-<br />
|tetR2:nsDNA -> nsDNA||1.93E-04||<br />
|-<br />
|Ø -> tetR2||1E-11||<br />
|-<br />
|aTc_ext -> aTc||3.3E-04||<br />
|-<br />
|gfp_mRNA -> Ø||1.16E-03||<br />
|-<br />
|gfp -> Ø||3.21E-05||<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||311000<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||311000<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||311000<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky3.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/Minnesota/22_June_2009Minnesota/22 June 20092009-07-29T19:46:34Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/19 June 2009|Go to Previous Day (June 19)]]'''|| width=158|'''[[Minnesota/23 June 2009|Go to Next Day (June 23)]]'''<br />
|}<br />
Here is the first incarnation of the TNN model (many future models will simply change this one):<br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
| RNAp + lacP + lacI4:lacO1 -> RNAp:lacP||6.23E+05|| <br />
|-<br />
| RNAp + lacP + lacO1 ↔ RNAp:lacP||1E+07|| 1<br />
|-<br />
|RNA:lacP -> RNAp:lacP* || .01 ||<br />
|-<br />
|RNAp:lacP* -> lacP + lacO1 + RNAp:DNAgfp||30||<br />
|-<br />
|RNAp:DNAgfp -> RNAp + gfp_mRNA||30||<br />
|-<br />
|gfp_mRNA + rib -> rib:gfp_mRNA||100000||<br />
|-<br />
|rib:gfp_mRNA -> rib:gfp_mRNA_1 + gfp_mRNA||33||<br />
|-<br />
|rib:gfp_mRNA_1 -> rib + gfp||33||<br />
|-<br />
|tetR2 + aTc ↔ tetR2:aTc||2E+09||4E-04<br />
|-<br />
|tetR2:aTc + aTc ↔ tetR2:aTc2||1E+08||1E-03<br />
|-<br />
|tetR2:aTc + tetO1 ↔ tetR2:tetO1:aTc||1E+08||1<br />
|-<br />
|tetR2:aTc2 + tetO1 ↔ tetR2:tetO1:aTc2||1E+08||1E+05<br />
|-<br />
|tetR2:tetO1 + aTc ↔ tetR2:tetO1:aTc||1E+08||1E-03<br />
|-<br />
|tetR2:tetO1:aTc + aTc ↔ tetR2:tetO1:aTc2||1E+08||1E-03<br />
|-<br />
|tetR2 -> Ø||2.89E-04||<br />
|-<br />
|tetR2:aTc -> aTc||2.89E-04||<br />
|-<br />
|tetR2:aTc2 -> 2 aTc||2.89E-04||<br />
|-<br />
|tetR2 + nsDNA ↔ tetR2:nsDNA||1000||3.2409<br />
|-<br />
|tetR2:aTc + nsDNA ↔ tetR2:aTc:nsDNA||1000||3.2409<br />
|-<br />
|tetR2:aTc:nsDNA -> aTc + nsDNA||1.93E-04||<br />
|-<br />
|tetR2:nsDNA -> nsDNA||1.93E-04||<br />
|-<br />
|Ø -> tetR2||1E-11||<br />
|-<br />
|Ø -> aTc||3.3E-04||<br />
|-<br />
|gfp_mRNA -> Ø||1.16E-03||<br />
|-<br />
|gfp -> Ø||3.21E-05||<br />
|}</div>Bswiniarhttp://2009.igem.org/Minnesota/24_July_2009Minnesota/24 July 20092009-07-29T19:42:15Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/23 July 2009|Go to Previous Day (July 23)]]'''|| width=158|'''[[Minnesota/27 July 2009|Go to Next Day (July 27)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnnew28.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
[[Image:Tnnnew30.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/Minnesota/23_July_2009Minnesota/23 July 20092009-07-29T19:42:02Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/22 July 2009|Go to Previous Day (July 22)]]'''|| width=158|'''[[Minnesota/24 July 2009|Go to Next Day (July 24)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnnew24.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
[[Image:Tnnnew26.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/Minnesota/22_July_2009Minnesota/22 July 20092009-07-29T19:41:45Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/21 July 2009|Go to Previous Day (July 21)]]'''|| width=158|'''[[Minnesota/23 July 2009|Go to Next Day (July 23)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnnew20.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
[[Image:Tnnnew22.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/File:Tnnnew30.jpgFile:Tnnnew30.jpg2009-07-29T19:41:16Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/File:Tnnnew28.jpgFile:Tnnnew28.jpg2009-07-29T19:41:09Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/File:Tnnnew26.jpgFile:Tnnnew26.jpg2009-07-29T19:41:00Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/File:Tnnnew24.jpgFile:Tnnnew24.jpg2009-07-29T19:40:53Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/File:Tnnnew22.jpgFile:Tnnnew22.jpg2009-07-29T19:40:45Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/File:Tnnnew20.jpgFile:Tnnnew20.jpg2009-07-29T19:40:36Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/File:Tnnnew18.jpgFile:Tnnnew18.jpg2009-07-29T19:40:27Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/Minnesota/21_July_2009Minnesota/21 July 20092009-07-29T19:40:12Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/20 July 2009|Go to Previous Day (July 20)]]'''|| width=158|'''[[Minnesota/22 July 2009|Go to Next Day (July 22)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnnew16.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
[[Image:Tnnnew18.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/Minnesota/21_July_2009Minnesota/21 July 20092009-07-29T19:40:00Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/20 July 2009|Go to Previous Day (July 20)]]'''|| width=158|'''[[Minnesota/22 July 2009|Go to Next Day (July 22)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnnew16.jpg.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
[[Image:Tnnnew18.jpg.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/File:Tnnnew16.jpgFile:Tnnnew16.jpg2009-07-29T19:39:39Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/Minnesota/21_July_2009Minnesota/21 July 20092009-07-29T19:39:18Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/20 July 2009|Go to Previous Day (July 20)]]'''|| width=158|'''[[Minnesota/22 July 2009|Go to Next Day (July 22)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky3.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
[[Image:Tnnatcvariesleaky3.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/File:Tnnatcvariesleaky35.jpgFile:Tnnatcvariesleaky35.jpg2009-07-29T19:38:31Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/Minnesota/20_July_2009Minnesota/20 July 20092009-07-29T19:38:23Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/17 July 2009|Go to Previous Day (July 17)]]'''|| width=158|'''[[Minnesota/21 July 2009|Go to Next Day (July 21)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky35.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/File:Tnnatcvariesleaky33.jpgFile:Tnnatcvariesleaky33.jpg2009-07-29T19:37:50Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/File:Tnnatcvariesleaky31.jpgFile:Tnnatcvariesleaky31.jpg2009-07-29T19:37:44Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/File:Tnnatcvariesleaky29.jpgFile:Tnnatcvariesleaky29.jpg2009-07-29T19:37:37Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/File:Tnnatcvariesleaky27.jpgFile:Tnnatcvariesleaky27.jpg2009-07-29T19:37:29Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/Minnesota/17_July_2009Minnesota/17 July 20092009-07-29T19:37:07Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/16 July 2009|Go to Previous Day (July 16)]]'''|| width=158|'''[[Minnesota/20 July 2009|Go to Next Day (July 20)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky33.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/Minnesota/16_July_2009Minnesota/16 July 20092009-07-29T19:36:58Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/15 July 2009|Go to Previous Day (July 15)]]'''|| width=158|'''[[Minnesota/17 July 2009|Go to Next Day (July 17)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky31.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/Minnesota/14_July_2009Minnesota/14 July 20092009-07-29T19:36:40Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/13 July 2009|Go to Previous Day (July 13)]]'''|| width=158|'''[[Minnesota/15 July 2009|Go to Next Day (July 15)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky29.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/Minnesota/13_July_2009Minnesota/13 July 20092009-07-29T19:36:29Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/10 July 2009|Go to Previous Day (July 10)]]'''|| width=158|'''[[Minnesota/14 July 2009|Go to Next Day (July 14)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky27.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/Minnesota/10_July_2009Minnesota/10 July 20092009-07-29T19:35:55Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/9 July 2009|Go to Previous Day (July 9)]]'''|| width=158|'''[[Minnesota/13 July 2009|Go to Next Day (July 13)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky25.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/Minnesota/8_July_2009Minnesota/8 July 20092009-07-29T19:35:44Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/7 July 2009|Go to Previous Day (July 7)]]'''|| width=158|'''[[Minnesota/9 July 2009|Go to Next Day (July 9)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky23.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniarhttp://2009.igem.org/File:Tnnatcvariesleaky25.jpgFile:Tnnatcvariesleaky25.jpg2009-07-29T19:35:26Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/File:Tnnatcvariesleaky23.jpgFile:Tnnatcvariesleaky23.jpg2009-07-29T19:35:19Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/File:Tnnatcvariesleaky21.jpgFile:Tnnatcvariesleaky21.jpg2009-07-29T19:35:13Z<p>Bswiniar: </p>
<hr />
<div></div>Bswiniarhttp://2009.igem.org/Minnesota/6_July_2009Minnesota/6 July 20092009-07-29T19:34:55Z<p>Bswiniar: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/3 July 2009|Go to Previous Day (July 3)]]'''|| width=158|'''[[Minnesota/7 July 2009|Go to Next Day (July 7)]]'''<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky21.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Bswiniar