http://2007.igem.org/wiki/index.php?title=Special:Contributions/Macampbell&feed=atom&limit=50&target=Macampbell&year=&month=2007.igem.org - User contributions [en]2024-03-28T13:12:22ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/File:Synthases.jpgFile:Synthases.jpg2007-12-12T17:07:58Z<p>Macampbell: </p>
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<div></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-10-27T04:32:38Z<p>Macampbell: /* Our Successful Project */</p>
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<div><center>[[Davidson Missouri W| <span style="color:black">Home</span>]] | [[Davidson Missouri W/Background Information| <span style="color:red">Background Information</span>]] | [[Davidson Missouri W/Solving the HPP in vivo| <span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]] | [[Davidson Missouri W/Mathematical Modeling| <span style="color:red">Mathematical Modeling</span>]] | [[Davidson Missouri W/Gene splitting| <span style="color:red">Gene Splitting</span>]] | [[Davidson Missouri W/Results| <span style="color:red">Results</span>]] | [[Davidson Missouri W/Traveling Salesperson Problem| <span style="color:red">Traveling Salesperson Problem</span> ]] | [[Davidson Missouri W/Software|<span style="color:red">Software</span>]] | [[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]</center><br />
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{| border="1" cellpadding="5" cellspacing="0" align="center" width="100%"<br />
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|style="color: black; background-color: red;" align="center"| '''Davidson'''<br />
<b><br />
[[Davidson Missouri W/Oyinade Adefuye|<span style="color:black">Oyinade Adefuye</span>]]<br />
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[[Davidson Missouri W/Will DeLoache|<span style="color:black">Will DeLoache</span>]]<br />
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[[Davidson Missouri W/Jim Dickson|<span style="color:black">Jim Dickson</span>]]<br />
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[[Davidson Missouri W/Andrew Martens|<span style="color:black">Andrew Martens</span>]]<br />
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[[Davidson Missouri W/Amber Shoecraft|<span style="color:black">Amber Shoecraft</span>]]<br />
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[[Davidson Missouri W/Mike Waters|<span style="color:black">Mike Waters</span>]]<br />
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[[Davidson Missouri W/A. Malcolm Campbell|<span style="color:black">A. Malcolm Campbell</span>]]<br />
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[[Davidson Missouri W/Karmella Haynes|<span style="color:black">Karmella Haynes</span>]]<br />
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[[Davidson Missouri W/Laurie Heyer|<span style="color:black">Laurie Heyer</span>]]<br />
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|style="color: black; background-color: gold;" align="center"|'''Missouri Western'''<br />
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[[Davidson Missouri W/Jordan Baumgardner|<span style="color:black;">Jordan Baumgardner</span>]]<br />
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[[Davidson Missouri W/Tom Crowley|<span style="color:black;">Tom Crowley</span>]]<br />
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[[Davidson Missouri W/Lane H. Heard|<span style="color:black;">Lane H. Heard</span>]]<br />
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[[Davidson Missouri W/Nickolaus Morton|<span style="color:black;">Nickolaus Morton</span>]]<br />
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[[Davidson Missouri W/Michelle Ritter|<span style="color:black;">Michelle Ritter</span>]]<br />
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[[Davidson Missouri W/Jessica Treece|<span style="color:black;">Jessica Treece</span>]]<br />
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[[Davidson Missouri W/Matthew Unzicker|<span style="color:black;">Matthew Unzicker</span>]]<br />
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[[Davidson Missouri W/Amanda Valencia|<span style="color:black;">Amanda Valencia</span>]]<br />
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[[Davidson Missouri W/Todd Eckdahl|<span style="color:black;">Todd Eckdahl</span>]]<br />
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[[Davidson Missouri W/Jeff Poet|<span style="color:black;">Jeff Poet</span>]]<br />
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=Our Successful Project=<br />
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{| border="1" cellpadding="5" cellspacing="0" align="center" width="90%"<br />
|-<br />
! style="color: black; background-color: red;" width="20%"| <font size="+1">In Depth</font><br />
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[[Davidson Missouri W/Background Information|<span style="color:red">Background Information</span>]]<br />
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[[Davidson Missouri W/Solving the HPP in vivo|<span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]]<br />
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[[Davidson Missouri W/Mathematical Modeling|<span style="color:red">Mathematical Modeling</span>]]<br />
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[[Davidson Missouri W/Gene splitting|<span style="color:red">Gene Splitting</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Results|<span style="color:red">Results</span>]]<br />
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[[Davidson Missouri W/Traveling Salesperson Problem|<span style="color:red">Traveling Salesperson Problem</span>]]<br />
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[[Davidson Missouri W/Software|<span style="color:red">Software</span>]]<br />
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[[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]<br />
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|Hamiltonian Path Problem<br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/''hix'' DNA recombination mechanism which exists in nature in ''Salmonella'' as standard biobricks for use in ''E. coli''. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we successfully continued our collaboration and our efforts to manipulate ''E. coli'' into mathematics problem solvers as we refine our efforts with the hin/''hix'' mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adleman in 1994 (see [[Davidson_Missouri_W/Resources_and_Citations | citations]]) where a unique Hamiltonian path was found ''in vitro'' for a particular directed graph on seven nodes. We were able to use bacterial computers to solve the Hamiltonian path problem ''in vivo''. ([[Davidson Missouri W/Background Information#Why Use Bacteria?|Why use a bacterial computer?]])<br />
<br />
<br> <br />
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[[Image:Adelman.png|thumb|300px|center|The Adleman graph.]] <br />
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<center> For the graph used in Adleman's paper (shown above), the Hamiltonian Path Problem would ask: can you find a path along the directed edges that travels from node 1 (green) to node 5 (red) and visits each node on the graph exactly once? <br><br />
[https://static.igem.org/mediawiki/2007/6/6f/Adelmansolution.png Click here] for the solution.<br />
</center><br />
|}<br />
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<br><br />
<center> '''A Human Representation of the Adleman Graph. (mouse over to see the full effect)'''<br />
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<Previous Section | [[Davidson Missouri W/Background Information | Next Section>]]<br />
</center></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-10-27T04:31:02Z<p>Macampbell: /* The Team */</p>
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<div><center>[[Davidson Missouri W| <span style="color:black">Home</span>]] | [[Davidson Missouri W/Background Information| <span style="color:red">Background Information</span>]] | [[Davidson Missouri W/Solving the HPP in vivo| <span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]] | [[Davidson Missouri W/Mathematical Modeling| <span style="color:red">Mathematical Modeling</span>]] | [[Davidson Missouri W/Gene splitting| <span style="color:red">Gene Splitting</span>]] | [[Davidson Missouri W/Results| <span style="color:red">Results</span>]] | [[Davidson Missouri W/Traveling Salesperson Problem| <span style="color:red">Traveling Salesperson Problem</span> ]] | [[Davidson Missouri W/Software|<span style="color:red">Software</span>]] | [[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]</center><br />
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{| border="1" cellpadding="5" cellspacing="0" align="center" width="100%"<br />
|-<br />
! style="color: white; background-color: black;"| The Team<br />
! style="color: white; background-color: black;" | The Faculty<br />
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|style="color: black; background-color: red;" align="center"| '''Davidson'''<br />
<b><br />
[[Davidson Missouri W/Oyinade Adefuye|<span style="color:black">Oyinade Adefuye</span>]]<br />
<br><br />
[[Davidson Missouri W/Will DeLoache|<span style="color:black">Will DeLoache</span>]]<br />
<br><br />
[[Davidson Missouri W/Jim Dickson|<span style="color:black">Jim Dickson</span>]]<br />
<br><br />
[[Davidson Missouri W/Andrew Martens|<span style="color:black">Andrew Martens</span>]]<br />
<br><br />
[[Davidson Missouri W/Amber Shoecraft|<span style="color:black">Amber Shoecraft</span>]]<br />
<br><br />
[[Davidson Missouri W/Mike Waters|<span style="color:black">Mike Waters</span>]]<br />
</b><br />
<br />
|style="color: black; background-color: red;" align="center"|<br />
<b><br />
[[Davidson Missouri W/A. Malcolm Campbell|<span style="color:black">A. Malcolm Campbell</span>]]<br />
<br><br />
[[Davidson Missouri W/Karmella Haynes|<span style="color:black">Karmella Haynes</span>]]<br />
<br><br />
[[Davidson Missouri W/Laurie Heyer|<span style="color:black">Laurie Heyer</span>]]<br />
</b><br />
<br />
|style="color: black; background-color: white;" align="center"|<br />
[[Image:DavidsonLogo.gif]]<br />
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|style="color: black; background-color: white;" align="center"|<br />
[[Image:Team1.jpg|thumb|center|300px]]<br />
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|-<br />
<br />
|style="color: black; background-color: gold;" align="center"|'''Missouri Western'''<br />
<b><br />
[[Davidson Missouri W/Jordan Baumgardner|<span style="color:black;">Jordan Baumgardner</span>]]<br />
<br><br />
[[Davidson Missouri W/Tom Crowley|<span style="color:black;">Tom Crowley</span>]]<br />
<br><br />
[[Davidson Missouri W/Lane H. Heard|<span style="color:black;">Lane H. Heard</span>]]<br />
<br><br />
[[Davidson Missouri W/Nickolaus Morton|<span style="color:black;">Nickolaus Morton</span>]]<br />
<br><br />
[[Davidson Missouri W/Michelle Ritter|<span style="color:black;">Michelle Ritter</span>]]<br />
<br><br />
[[Davidson Missouri W/Jessica Treece|<span style="color:black;">Jessica Treece</span>]]<br />
<br><br />
[[Davidson Missouri W/Matthew Unzicker|<span style="color:black;">Matthew Unzicker</span>]]<br />
<br><br />
[[Davidson Missouri W/Amanda Valencia|<span style="color:black;">Amanda Valencia</span>]]<br />
</b><br />
<br />
|style="color: black; background-color: gold;" align="center"|<br />
<b><br />
[[Davidson Missouri W/Todd Eckdahl|<span style="color:black;">Todd Eckdahl</span>]]<br />
<br><br />
[[Davidson Missouri W/Jeff Poet|<span style="color:black;">Jeff Poet</span>]]<br />
</b><br />
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|style="color: black; background-color: white;" align="center"|[[Image:MWLogo.gif]]<br />
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|style="color: black; background-color: white;" align="center"|[[Image:MWSUteam.jpeg|thumb|center|300px]]<br />
|-<br />
<br />
|}<br />
<br><br />
<center><br />
<br />
=Our Successful Project=<br />
</center><br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center" width="90%"<br />
|-<br />
! style="color: black; background-color: red;" width="20%"| <font size="+1">In Depth</font><br />
! colspan="3" style="color: black; background-color: red;" width="60%"| <font size="+1">Overview</font><br />
|-<br />
|style="color: black; background-color: black;" align="center"|<br />
[[Davidson Missouri W/Background Information|<span style="color:red">Background Information</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Solving the HPP in vivo|<span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Mathematical Modeling|<span style="color:red">Mathematical Modeling</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Gene splitting|<span style="color:red">Gene Splitting</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Results|<span style="color:red">Results</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Traveling Salesperson Problem|<span style="color:red">Traveling Salesperson Problem</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Software|<span style="color:red">Software</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]<br />
<br><br><Br><br />
|Hamiltonian Path Problem<br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/''hix'' DNA recombination mechanism which exists in nature in ''Salmonella'' as standard biobricks for use in ''E. coli''. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we successfully continued our collaboration and our efforts to manipulate ''E. coli'' into mathematics problem solvers as we refine our efforts with the hin/''hix'' mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adleman in 1994 (see [[Davidson_Missouri_W/Resources_and_Citations | citations]]) where a unique Hamiltonian path was found ''in vitro'' for a particular directed graph on seven nodes. We were able to use bacterial computers to solve the Hamiltonian path problem ''in vivo''. ([[Davidson Missouri W/Background Information#Why Use Bacteria?|Why use a bacterial computer?]])<br />
<br />
<br> <br />
<br />
[[Image:Adelman.png|thumb|300px|center|The Adleman graph.]] <br />
<br />
<center> For the graph used in Adleman's paper (shown above), the Hamiltonian Path Problem would ask: can you find a path along the directed edges that travels from node 1 (green) to node 5 (red) and visits each node on the graph exactly once? <br><br />
[https://static.igem.org/mediawiki/2007/6/6f/Adelmansolution.png Click here] for the solution.<br />
</center><br />
|}<br />
<br />
<br><br />
<center> '''A Human Representation of the Adleman Graph.'''<br />
<br />
<html><br />
<head><br />
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<Previous Section | [[Davidson Missouri W/Background Information | Next Section>]]<br />
</center></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_W/ResultsDavidson Missouri W/Results2007-10-27T01:22:09Z<p>Macampbell: /* Conclusions */</p>
<hr />
<div><center>[[Davidson Missouri W| <span style="color:red">Home</span>]] | [[Davidson Missouri W/Background Information| <span style="color:red">Background Information</span>]] | [[Davidson Missouri W/Solving the HPP in vivo| <span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]] | [[Davidson Missouri W/Mathematical Modeling| <span style="color:red">Mathematical Modeling</span>]] | [[Davidson Missouri W/Gene splitting| <span style="color:red">Gene Splitting</span>]] | [[Davidson Missouri W/Results| <span style="color:black">Results</span>]] | [[Davidson Missouri W/Traveling Salesperson Problem| <span style="color:red">Traveling Salesperson Problem</span> ]] | [[Davidson Missouri W/Software|<span style="color:red">Software</span>]] | [[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]</center><br />
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<br />
==Gene Splitting Worked==<br />
We were able to split two genes: GFP and RFP. Split GFP has strong green fluorescence. Split RFP's red color is much reduced compared to wild-type RFP. It takes overnight incubation at room temperature for the red color to be visible in white light. Both colors are fluorescent under UV light, although the green color predominates. In [https://2007.igem.org/Image:GFPplates.jpg Figure 1] below, a negative-control (on the left) does not fluoresce, but split GFP (on the right) does. [https://2007.igem.org/Image:Split_RFP.png Figure 2] shows a plate of cells containing split RFP.<br />
<br />
[[Image:GFPplates.jpg|thumb|left|500px|'''Figure 1''': The negative control (hixC-GFP2) on the left does not fluoresce, but the experimental split GFP (pLac-RBS-GFP1-hixC-GFP2) on the right does.]]<br />
<br />
[[Image:Split_RFP.png|thumb|left|250px|'''Figure 2''': Cells containing split RFP (pLac-RBS-RFP1-hixC-RFP2) after overnight incubation at room temperature.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Graphs==<br />
<br />
Once we managed to split two genes, we proceeded to implement two different 3-node graphs into plasmids. These graphs were named Graphs A and B and are shown below. For each graph, we wanted to find a Hamiltonian Path from the node represented by RFP (colored in green) to the node represented by the transcriptional terminator (colored in red). The plasmid representation of Graph A is not be capable of flipping into a [https://2007.igem.org/Davidson_Missouri_W/Mathematical_Modeling#True_Positives_and_False_Positives false positive] orientation, while the plasmid representation of Graph B does provide that possibility.<br />
<br />
[[Image:MWSUgraph.png|thumb|200px|left|'''Graph A''': This graph contains 3-nodes and 3-edges. We wanted to find a Hamiltonian path from the node representing RFP (colored in green) to the node representing the transcriptional terminator (colored in red).]] [[Image:DCgraph.png|thumb|200px|none|'''Graph B''': This graph also contains 3-nodes and 3-edges, however it provides the possibility of a false positive solution.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Starting Orientations==<br />
We constructed the following Hamiltonian Path Problem (HPP) constructs to test our bacterial computer system for Graphs A and B. Constructs that are built to solve [https://2007.igem.org/Image:MWSUgraph.png Graph A] are labeled HPP-A. Constructs that are built to solve [https://2007.igem.org/Image:DCgraph.png Graph B] are labeled HPP-B. A subscript equal to 0 denotes a plasmid in a solved orientation. A subscript that is greater than 0 denotes a plasmid that is in an unordered starting orientation.<br />
<br />
*'''HPP<sub>0</sub> (Positive Control for HPP-A and HPP-B constructs) '''<br>[[Image:Picture_1.png]]<br><br />
*'''HPP-A<sub>0</sub> (Solved Orientation)'''<br>[[Image:Picture_2.png]]<br><br />
*'''HPP-A<sub>1</sub> '''<br>[[Image:Picture_3.png]]<br><br />
*'''HPP-A<sub>2</sub> '''<br>[[Image:Picture_4.png]]<br><br />
*'''HPP-B<sub>1</sub> '''<br>[[Image:Picture_5.png]]<br><br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Controls==<br />
Because are graphs require that solved colonies be selected for by double fluorescence (both red and green), we first performed control experiments to ensure that we could distinguish solved and unsolved colonies. To test fluorescence phenotypes when both split GFP and split RFP are the same cell, we used our positive control constructs ([http://partsregistry.org/Part:BBa_I715045 HPP<sub>0</sub>] and [http://partsregistry.org/Part:BBa_I715042 HPP-A<sub>0</sub>]) with split RFP and split GFP both downstream of the T7 polymerase promoter. In the presence of T7 polymerase, cells with the split RFP/GFP plasmid should demonstrate both green and red fluorescence. <br />
<br />
We first tried cotransforming our HPP<sub>0</sub> construct with a T7 RNA polymerase plasmid construct ([http://partsregistry.org/Part:BBa_I715038 BBa_I715038]). As can be seen in [https://2007.igem.org/Image:SplitRFP-GFP.jpg Figure 3] below, this cotransformation resulted in bright green colonies that demonstrated minimal red fluorescence. When we measured the fluorescence of the cotransformed control, RFP was not present at detectable levels (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
We then tried transforming our HPP-A<sub>0</sub> control plasmid into cells that already contained T7 polymerase in their chromosome (T7 RNAP cells). As can be seen in [https://2007.igem.org/Image:DSC02737.jpeg Figure 4] below, these colonies displayed the yellowish color we expected. Based on our fluorescence data, (shown [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]), we were able to show that GFP and RFP were present at detectable levels in this control. We hypothesize that the difference in the two controls is due to higher concentrations of T7 RNA polymerase in the T7 RNAP cells than in the cotransformants. We therefore used the T7 RNAP cells for future experiments.<br />
<br />
[[Image:SplitRFP-GFP.jpg|thumb|left|300px|'''Figure 3''': Colonies containing both the T7 RNA polymerase plasmid and the HPP<sub>0</sub> control plasmid demonstrated strong green fluorescence, but minimal red fluorescence.]]<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|380px|'''Figure 4''': T7 RNAP colonies that contain the HPP-A<sub>0</sub> control plasmid demonstrated the expected yellow fluorescence.]]<br />
[[Image:Linebreak.png]]<br />
<br />
In addition to the regular split RFP/GFP controls, we also tested 2 "hybrid" constructs to ensure that they would not fluoresce. GFP and RFP have similar structures and functions. Previous studies have shown that it is possible to modify GFP to display a wide range of color phenotypes. We created the following parts: Plac-RBS-RFP1-hixC-GFP2 ([http://partsregistry.org/Part:BBa_I715036 BBa_I715036]) and Plac-RBS-GFP1-hixC-RFP2 ([http://partsregistry.org/Part:BBa_I715035 BBa_I715035]). A plasmid may, at some point during its flipping process, contain such sequences. We tested to make sure that the similarity of these two proteins did not make them compatible enough to fluoresce. It was found that neither of these parts show any fluorescence or color change (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Dectection of Fluoresence by Spectroscopy==<br />
We used a fluorometer to measure the fluorescence of various HPP constructs. First, we had to determine the excitation and emission wavelengths that would allow us to detect the green and red fluorescence of split genes. Scans were conducted in order to find these wavelengths. The graphs below show scans of excitation wavelengths that produce fluorescence at an emission wavelength of 515 nm , the wavelength we found to be best for green, and 608 nm, the best wavelength for red. We used these scans to pick a peak emission wavelength for further testing. <br />
<br />
For these scans we used the following constructs:<br><br />
'''pLac-RBS-GFP1-''hixC''-GFP2''' - This construct fluoresces green on the UV box.<br><br />
'''HPP-A<sub>0</sub>''' - This construct fluoresces yellow on the UV box.<br><br />
'''HPP-A<sub>1</sub>''' - This construct fluoresces red on the UV box.<br><br />
'''HPP-A<sub>2</sub>''' - This construct shows no fluorescence on the UV box.<br><br />
<br><br />
<br />
[[Image:GFP Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 515 nm (green light)]]<br />
<br />
[[Image:Red Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 608 nm (red light)]]<br />
<br />
<br><br />
On the basis of these scans, we used excitation/emission wavelengths of 450 nm / 515 nm for green and 560 nm / 608 nm for red to obtain the results shown below.<br />
<br><br />
<br />
===<center>Normalized* Fluorimetry Results</center>===<br />
{| border="1" cellpadding="5" cellspacing="0" align="center" width="100%"<br />
|-<br />
! style="color: black; background-color: white;" | Construct<br />
! style="color: black; background-color: white;" | Fluorescent Color on UV Box<br />
! style="color: black; background-color: white;" | Green<br />
! style="color: black; background-color: white;" | Red<br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span><br />
|Red<br />
|7<br />
|<span style="color:red">263</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span>-RBS-<span style="color:green">GFP</span><br />
|Red<br />
|<span style="color:green">144</span><br />
|<span style="color:red">370</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|Green<br />
|<span style="color:green">136</span><br />
|0<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|0<br />
|<span style="color:red">147</span><br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|11<br />
|2<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|None<br />
|13<br />
|2<br />
|-<br />
<br />
|HPP<sub>0</sub>***<br />
|Green<br />
|<span style="color:green">72</span><br />
|18<br />
|-<br />
<br />
|HPP-A<sub>0</sub><br />
|Yellow<br />
|<span style="color:green">340</span><br />
|<span style="color:red">255</span><br />
|-<br />
<br />
|HPP-A<sub>1</sub><br />
|Red<br />
|1<br />
|<span style="color:red">143</span><br />
|-<br />
<br />
|HPP-A<sub>2</sub><br />
|None<br />
|11<br />
|3<br />
|-<br />
<br />
<br />
|HPP-B<sub>1</sub>***<br />
|hybrid green<br />
|15<br />
|3<br />
|-<br />
<br />
|}<br />
'* To normalize the data, 57 fluorescence units were subtracted from the Green column and 18 from the Red column. These were the lowest fluorescence values for each wavelength.<br><br />
'*** These 2 constructs were cotransformed with the T7 RNA polymerase plasmid (as opposed to being transformed into T7 RNAP cells). This could explain their reduced fluorescence.<br />
<br />
<br><br />
<br />
==Solution Detected by Phenotype==<br />
<br />
To test for flipping by phenotype, we used our 3 - [[Davidson_Missouri_W/Results#Constructs |Graph A]] constructs from above, HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>. As expected, unflipped HPP-A<sub>0</sub> fluoresced yellow, unflipped HPP-A<sub>1</sub> fluoresced red, and unflipped HPP-A<sub>2</sub> demonstrated no fluorescence when transformed into the T7 RNAP cells (See Figures 5-7 below).<br />
<br />
A fragment containing the Hin expression cassette (pLac-RBS-Hin-LVA, [http://partsregistry.org/Part:BBa_S03536 BBa_S03536]) was ligated in front of each of the 3 HPP-A constructs. These HPP-A + Hin plasmids were then transformed into T7 RNAP cells. A colony from each transformation was picked and grown overnight for plasmid mini-prep. Each of the 3 constructs was restriction digested and the insert sizes were verified to be correct. The 3 constructs were then retransformed into T7 RNAP cells, and the resulting transformation mixture was streaked for colony isolation. '''The HPP-A<sub>0</sub> + Hin plate contained mostly yellow colonies but also red and green (See Figures 8 and 11). The Hin HPP-A<sub>1</sub> + Hin plate contained red, green and yellow colonies (See Figures 9 and 12). The HPP-A<sub>2</sub> + Hin plate contained mostly nonfluorescent colonies with a few green and red colonies (See Figures 10 and 13).'''<br><br />
<br />
<br />
''Click twice on the images below for higher resolution.''<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|270px|'''Figure 5:''' HPP-A<sub>0</sub> - These colonies fluoresced yellow]] [[Image:DSC02738.jpeg|thumb|left|270px|'''Figure 6:''' HPP-A<sub>1</sub> - These colonies fluoresced red]] [[Image:DSC02739.jpeg|thumb|left|270px|'''Figure 7:''' HPP-A<sub>2</sub> - These colonies do not fluoresce]]<br />
<br />
[[Image:DSC02746.jpeg|thumb|left|270px|'''Figure 8:''' HPP-A<sub>0</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02744.jpeg|thumb|left|270px|'''Figure 9:''' HPP-A<sub>1</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02736.jpeg|thumb|left|270px|'''Figure 10:''' HPP-A<sub>2</sub> + Hin - The addition of Hin has produced green and red colonies]]<br />
<br />
[[Image:Hin-ABC-cropped2.JPG|thumb|left|270px|'''Figure 11:''' HPP-A<sub>0</sub> + Hin - closeup of Figure 8]] <br />
[[Image:Hin-ACB-cropped3.JPG|thumb|left|270px|'''Figure 12:''' HPP-A<sub>1</sub> + Hin - closeup of Figure 9]]<br />
[[Image:Hin-BAC-cropped.JPG|thumb|left|270px|'''Figure 13:''' HPP-A<sub>2</sub> + Hin - closeup of Figure 10]]<br />
[[Image:Linebreak.png]]<br />
[[Image:Hin-ABC-high.jpg|thumb|left|270px|'''Figure 14:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-high.jpg|thumb|left|270px|'''Figure 15:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-high.jpg|thumb|left|270px|'''Figure 16:''' HPP-A<sub>2</sub> + Hin]]<br />
[[Image:Hin-ABC-low.jpg|thumb|left|270px|'''Figure 16:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-low.jpg|thumb|left|270px|'''Figure 17:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-low.jpg|thumb|left|270px|'''Figure 18:''' HPP-A<sub>2</sub> + Hin]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Solution Detected by PCR==<br />
<br />
In order to provide physical evidence of Hin-mediated DNA rearrangement of our HPP-A + Hin constructs, we designed PCR primers that would allow us to determine the position of the GFP2 gene half. The experiment below shows the results of PCR using the following multiplex primers: the universal reverse primer [http://partsregistry.org/Part:BBa_G00101 G00101], which binds to the 3' biobrick suffix, a forward primer that binds to RFP1, and a GFP2-forward primer. <br><br><br />
Here is how the primers bind to the 3 HPP-A constructs:<br><br />
[[Image:PCR with Primers.jpeg.png|frame|none|400px|'''PCR Primer Binding''': (from top to bottom) HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>]]<br><br />
<br><br />
[[Image:PCR 10-25-07.JPG|thumb|left|270px|Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lanes 2 : HPP-A<sub>0</sub>, Lanes 3 : HPP-A<sub>1</sub>, Lanes 4 : HPP-A<sub>2</sub>, Lanes 5 : Hin+HPP-A<sub>0</sub>, Lanes 6 : Hin+HPP-A<sub>1</sub>, Lanes 7 : Hin+HPP-A<sub>2</sub>]]<br />
<br><br />
[[Image:gels_10_25_07.JPG|thumb|left|270px|'''Left Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lanes 2,3 : ClaI digest of Hin+HPP-A<sub>0</sub>, Lanes 4,5 : ClaI digest of Hin+HPP-A<sub>1</sub>, Lanes 6,7 : ClaI digest of Hin+HPP-A<sub>2</sub><br><br />
'''Right Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lane 2 : unrelated digest, Lane 3 : EcoRI+PstI digest of Hin+HPP-A<sub>0</sub>, Lane 4 : unrelated digest, Lane 5 : EcoRI+PstI digest of Hin+HPP-A<sub>1</sub>, Lane 6 : unrelated digest, Lane 7 : EcoRI+PstI digest of Hin+HPP-A<sub>2</sub>]]<br />
<br />
<br />
<br><br />
[[Image:Linebreak.png]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Conclusions==<br />
We were able to insert a 13 amino acid ''hixC'' site into Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), maintaining the functionality of the proteins and allowing for flipping of the DNA by Hin Recombinase. When both split RFP and split GFP are present in the same cell, a yellow color can be observed by eye under UV light, and both red and green fluorescence are detected by fluorimetry. We then constructed a bacterial computer capable of solving a 3-node Hamiltonian Path Problem. We detected the solution by isolating cells that fluoresced yellow (both red and green).<br />
<br />
To test for flipping of our HPP computer constructs, we attatched a Hin-expression vector to the HPP construct and observed phenotypic differences in colonies. For all of the starting orientations, we observed colonies of varying colors, suggesting that some colonies had flipped away from their starting orientation and into a new orientation that produced a different phenotype. We also tested for flipping by PCR and were able to see multiple bands for all of our flipped constructs. These results indicate that flipping has occured in each of our HPP constructs. <br />
<br />
In the coming days, we plan to retransform our flipped HPP plasmid (with Hin-Recombinase removed from the plasmid) into T7 RNAP cells in order to obtain clonal colonies. We will screen for yellow colonies and sequencing any plasmids to verify correct solutions to the Hamiltonian Path Problem and solved by ''E. coli''. <br />
<br />
<br><br />
<hr><br />
<center><br />
<[[Davidson Missouri W/Gene splitting | Previous Section]] | [[Davidson Missouri W/Traveling Salesperson Problem | Next Section>]]<br />
</center></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_W/ResultsDavidson Missouri W/Results2007-10-27T01:18:16Z<p>Macampbell: /* Flipping Detection by PCR */</p>
<hr />
<div><center>[[Davidson Missouri W| <span style="color:red">Home</span>]] | [[Davidson Missouri W/Background Information| <span style="color:red">Background Information</span>]] | [[Davidson Missouri W/Solving the HPP in vivo| <span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]] | [[Davidson Missouri W/Mathematical Modeling| <span style="color:red">Mathematical Modeling</span>]] | [[Davidson Missouri W/Gene splitting| <span style="color:red">Gene Splitting</span>]] | [[Davidson Missouri W/Results| <span style="color:black">Results</span>]] | [[Davidson Missouri W/Traveling Salesperson Problem| <span style="color:red">Traveling Salesperson Problem</span> ]] | [[Davidson Missouri W/Software|<span style="color:red">Software</span>]] | [[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]</center><br />
<br />
<hr><br />
<br><br />
<br />
==Gene Splitting Worked==<br />
We were able to split two genes: GFP and RFP. Split GFP has strong green fluorescence. Split RFP's red color is much reduced compared to wild-type RFP. It takes overnight incubation at room temperature for the red color to be visible in white light. Both colors are fluorescent under UV light, although the green color predominates. In [https://2007.igem.org/Image:GFPplates.jpg Figure 1] below, a negative-control (on the left) does not fluoresce, but split GFP (on the right) does. [https://2007.igem.org/Image:Split_RFP.png Figure 2] shows a plate of cells containing split RFP.<br />
<br />
[[Image:GFPplates.jpg|thumb|left|500px|'''Figure 1''': The negative control (hixC-GFP2) on the left does not fluoresce, but the experimental split GFP (pLac-RBS-GFP1-hixC-GFP2) on the right does.]]<br />
<br />
[[Image:Split_RFP.png|thumb|left|250px|'''Figure 2''': Cells containing split RFP (pLac-RBS-RFP1-hixC-RFP2) after overnight incubation at room temperature.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Graphs==<br />
<br />
Once we managed to split two genes, we proceeded to implement two different 3-node graphs into plasmids. These graphs were named Graphs A and B and are shown below. For each graph, we wanted to find a Hamiltonian Path from the node represented by RFP (colored in green) to the node represented by the transcriptional terminator (colored in red). The plasmid representation of Graph A is not be capable of flipping into a [https://2007.igem.org/Davidson_Missouri_W/Mathematical_Modeling#True_Positives_and_False_Positives false positive] orientation, while the plasmid representation of Graph B does provide that possibility.<br />
<br />
[[Image:MWSUgraph.png|thumb|200px|left|'''Graph A''': This graph contains 3-nodes and 3-edges. We wanted to find a Hamiltonian path from the node representing RFP (colored in green) to the node representing the transcriptional terminator (colored in red).]] [[Image:DCgraph.png|thumb|200px|none|'''Graph B''': This graph also contains 3-nodes and 3-edges, however it provides the possibility of a false positive solution.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Starting Orientations==<br />
We constructed the following Hamiltonian Path Problem (HPP) constructs to test our bacterial computer system for Graphs A and B. Constructs that are built to solve [https://2007.igem.org/Image:MWSUgraph.png Graph A] are labeled HPP-A. Constructs that are built to solve [https://2007.igem.org/Image:DCgraph.png Graph B] are labeled HPP-B. A subscript equal to 0 denotes a plasmid in a solved orientation. A subscript that is greater than 0 denotes a plasmid that is in an unordered starting orientation.<br />
<br />
*'''HPP<sub>0</sub> (Positive Control for HPP-A and HPP-B constructs) '''<br>[[Image:Picture_1.png]]<br><br />
*'''HPP-A<sub>0</sub> (Solved Orientation)'''<br>[[Image:Picture_2.png]]<br><br />
*'''HPP-A<sub>1</sub> '''<br>[[Image:Picture_3.png]]<br><br />
*'''HPP-A<sub>2</sub> '''<br>[[Image:Picture_4.png]]<br><br />
*'''HPP-B<sub>1</sub> '''<br>[[Image:Picture_5.png]]<br><br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Controls==<br />
Because are graphs require that solved colonies be selected for by double fluorescence (both red and green), we first performed control experiments to ensure that we could distinguish solved and unsolved colonies. To test fluorescence phenotypes when both split GFP and split RFP are the same cell, we used our positive control constructs ([http://partsregistry.org/Part:BBa_I715045 HPP<sub>0</sub>] and [http://partsregistry.org/Part:BBa_I715042 HPP-A<sub>0</sub>]) with split RFP and split GFP both downstream of the T7 polymerase promoter. In the presence of T7 polymerase, cells with the split RFP/GFP plasmid should demonstrate both green and red fluorescence. <br />
<br />
We first tried cotransforming our HPP<sub>0</sub> construct with a T7 RNA polymerase plasmid construct ([http://partsregistry.org/Part:BBa_I715038 BBa_I715038]). As can be seen in [https://2007.igem.org/Image:SplitRFP-GFP.jpg Figure 3] below, this cotransformation resulted in bright green colonies that demonstrated minimal red fluorescence. When we measured the fluorescence of the cotransformed control, RFP was not present at detectable levels (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
We then tried transforming our HPP-A<sub>0</sub> control plasmid into cells that already contained T7 polymerase in their chromosome (T7 RNAP cells). As can be seen in [https://2007.igem.org/Image:DSC02737.jpeg Figure 4] below, these colonies displayed the yellowish color we expected. Based on our fluorescence data, (shown [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]), we were able to show that GFP and RFP were present at detectable levels in this control. We hypothesize that the difference in the two controls is due to higher concentrations of T7 RNA polymerase in the T7 RNAP cells than in the cotransformants. We therefore used the T7 RNAP cells for future experiments.<br />
<br />
[[Image:SplitRFP-GFP.jpg|thumb|left|300px|'''Figure 3''': Colonies containing both the T7 RNA polymerase plasmid and the HPP<sub>0</sub> control plasmid demonstrated strong green fluorescence, but minimal red fluorescence.]]<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|380px|'''Figure 4''': T7 RNAP colonies that contain the HPP-A<sub>0</sub> control plasmid demonstrated the expected yellow fluorescence.]]<br />
[[Image:Linebreak.png]]<br />
<br />
In addition to the regular split RFP/GFP controls, we also tested 2 "hybrid" constructs to ensure that they would not fluoresce. GFP and RFP have similar structures and functions. Previous studies have shown that it is possible to modify GFP to display a wide range of color phenotypes. We created the following parts: Plac-RBS-RFP1-hixC-GFP2 ([http://partsregistry.org/Part:BBa_I715036 BBa_I715036]) and Plac-RBS-GFP1-hixC-RFP2 ([http://partsregistry.org/Part:BBa_I715035 BBa_I715035]). A plasmid may, at some point during its flipping process, contain such sequences. We tested to make sure that the similarity of these two proteins did not make them compatible enough to fluoresce. It was found that neither of these parts show any fluorescence or color change (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Dectection of Fluoresence by Spectroscopy==<br />
We used a fluorometer to measure the fluorescence of various HPP constructs. First, we had to determine the excitation and emission wavelengths that would allow us to detect the green and red fluorescence of split genes. Scans were conducted in order to find these wavelengths. The graphs below show scans of excitation wavelengths that produce fluorescence at an emission wavelength of 515 nm , the wavelength we found to be best for green, and 608 nm, the best wavelength for red. We used these scans to pick a peak emission wavelength for further testing. <br />
<br />
For these scans we used the following constructs:<br><br />
'''pLac-RBS-GFP1-''hixC''-GFP2''' - This construct fluoresces green on the UV box.<br><br />
'''HPP-A<sub>0</sub>''' - This construct fluoresces yellow on the UV box.<br><br />
'''HPP-A<sub>1</sub>''' - This construct fluoresces red on the UV box.<br><br />
'''HPP-A<sub>2</sub>''' - This construct shows no fluorescence on the UV box.<br><br />
<br><br />
<br />
[[Image:GFP Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 515 nm (green light)]]<br />
<br />
[[Image:Red Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 608 nm (red light)]]<br />
<br />
<br><br />
On the basis of these scans, we used excitation/emission wavelengths of 450 nm / 515 nm for green and 560 nm / 608 nm for red to obtain the results shown below.<br />
<br><br />
<br />
===<center>Normalized* Fluorimetry Results</center>===<br />
{| border="1" cellpadding="5" cellspacing="0" align="center" width="100%"<br />
|-<br />
! style="color: black; background-color: white;" | Construct<br />
! style="color: black; background-color: white;" | Fluorescent Color on UV Box<br />
! style="color: black; background-color: white;" | Green<br />
! style="color: black; background-color: white;" | Red<br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span><br />
|Red<br />
|7<br />
|<span style="color:red">263</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span>-RBS-<span style="color:green">GFP</span><br />
|Red<br />
|<span style="color:green">144</span><br />
|<span style="color:red">370</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|Green<br />
|<span style="color:green">136</span><br />
|0<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|0<br />
|<span style="color:red">147</span><br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|11<br />
|2<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|None<br />
|13<br />
|2<br />
|-<br />
<br />
|HPP<sub>0</sub>***<br />
|Green<br />
|<span style="color:green">72</span><br />
|18<br />
|-<br />
<br />
|HPP-A<sub>0</sub><br />
|Yellow<br />
|<span style="color:green">340</span><br />
|<span style="color:red">255</span><br />
|-<br />
<br />
|HPP-A<sub>1</sub><br />
|Red<br />
|1<br />
|<span style="color:red">143</span><br />
|-<br />
<br />
|HPP-A<sub>2</sub><br />
|None<br />
|11<br />
|3<br />
|-<br />
<br />
<br />
|HPP-B<sub>1</sub>***<br />
|hybrid green<br />
|15<br />
|3<br />
|-<br />
<br />
|}<br />
'* To normalize the data, 57 fluorescence units were subtracted from the Green column and 18 from the Red column. These were the lowest fluorescence values for each wavelength.<br><br />
'*** These 2 constructs were cotransformed with the T7 RNA polymerase plasmid (as opposed to being transformed into T7 RNAP cells). This could explain their reduced fluorescence.<br />
<br />
<br><br />
<br />
==Solution Detected by Phenotype==<br />
<br />
To test for flipping by phenotype, we used our 3 - [[Davidson_Missouri_W/Results#Constructs |Graph A]] constructs from above, HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>. As expected, unflipped HPP-A<sub>0</sub> fluoresced yellow, unflipped HPP-A<sub>1</sub> fluoresced red, and unflipped HPP-A<sub>2</sub> demonstrated no fluorescence when transformed into the T7 RNAP cells (See Figures 5-7 below).<br />
<br />
A fragment containing the Hin expression cassette (pLac-RBS-Hin-LVA, [http://partsregistry.org/Part:BBa_S03536 BBa_S03536]) was ligated in front of each of the 3 HPP-A constructs. These HPP-A + Hin plasmids were then transformed into T7 RNAP cells. A colony from each transformation was picked and grown overnight for plasmid mini-prep. Each of the 3 constructs was restriction digested and the insert sizes were verified to be correct. The 3 constructs were then retransformed into T7 RNAP cells, and the resulting transformation mixture was streaked for colony isolation. '''The HPP-A<sub>0</sub> + Hin plate contained mostly yellow colonies but also red and green (See Figures 8 and 11). The Hin HPP-A<sub>1</sub> + Hin plate contained red, green and yellow colonies (See Figures 9 and 12). The HPP-A<sub>2</sub> + Hin plate contained mostly nonfluorescent colonies with a few green and red colonies (See Figures 10 and 13).'''<br><br />
<br />
<br />
''Click twice on the images below for higher resolution.''<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|270px|'''Figure 5:''' HPP-A<sub>0</sub> - These colonies fluoresced yellow]] [[Image:DSC02738.jpeg|thumb|left|270px|'''Figure 6:''' HPP-A<sub>1</sub> - These colonies fluoresced red]] [[Image:DSC02739.jpeg|thumb|left|270px|'''Figure 7:''' HPP-A<sub>2</sub> - These colonies do not fluoresce]]<br />
<br />
[[Image:DSC02746.jpeg|thumb|left|270px|'''Figure 8:''' HPP-A<sub>0</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02744.jpeg|thumb|left|270px|'''Figure 9:''' HPP-A<sub>1</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02736.jpeg|thumb|left|270px|'''Figure 10:''' HPP-A<sub>2</sub> + Hin - The addition of Hin has produced green and red colonies]]<br />
<br />
[[Image:Hin-ABC-cropped2.JPG|thumb|left|270px|'''Figure 11:''' HPP-A<sub>0</sub> + Hin - closeup of Figure 8]] <br />
[[Image:Hin-ACB-cropped3.JPG|thumb|left|270px|'''Figure 12:''' HPP-A<sub>1</sub> + Hin - closeup of Figure 9]]<br />
[[Image:Hin-BAC-cropped.JPG|thumb|left|270px|'''Figure 13:''' HPP-A<sub>2</sub> + Hin - closeup of Figure 10]]<br />
[[Image:Linebreak.png]]<br />
[[Image:Hin-ABC-high.jpg|thumb|left|270px|'''Figure 14:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-high.jpg|thumb|left|270px|'''Figure 15:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-high.jpg|thumb|left|270px|'''Figure 16:''' HPP-A<sub>2</sub> + Hin]]<br />
[[Image:Hin-ABC-low.jpg|thumb|left|270px|'''Figure 16:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-low.jpg|thumb|left|270px|'''Figure 17:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-low.jpg|thumb|left|270px|'''Figure 18:''' HPP-A<sub>2</sub> + Hin]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Solution Detected by PCR==<br />
<br />
In order to provide physical evidence of Hin-mediated DNA rearrangement of our HPP-A + Hin constructs, we designed PCR primers that would allow us to determine the position of the GFP2 gene half. The experiment below shows the results of PCR using the following multiplex primers: the universal reverse primer [http://partsregistry.org/Part:BBa_G00101 G00101], which binds to the 3' biobrick suffix, a forward primer that binds to RFP1, and a GFP2-forward primer. <br><br><br />
Here is how the primers bind to the 3 HPP-A constructs:<br><br />
[[Image:PCR with Primers.jpeg.png|frame|none|400px|'''PCR Primer Binding''': (from top to bottom) HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>]]<br><br />
<br><br />
[[Image:PCR 10-25-07.JPG|thumb|left|270px|Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lanes 2 : HPP-A<sub>0</sub>, Lanes 3 : HPP-A<sub>1</sub>, Lanes 4 : HPP-A<sub>2</sub>, Lanes 5 : Hin+HPP-A<sub>0</sub>, Lanes 6 : Hin+HPP-A<sub>1</sub>, Lanes 7 : Hin+HPP-A<sub>2</sub>]]<br />
<br><br />
[[Image:gels_10_25_07.JPG|thumb|left|270px|'''Left Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lanes 2,3 : ClaI digest of Hin+HPP-A<sub>0</sub>, Lanes 4,5 : ClaI digest of Hin+HPP-A<sub>1</sub>, Lanes 6,7 : ClaI digest of Hin+HPP-A<sub>2</sub><br><br />
'''Right Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lane 2 : unrelated digest, Lane 3 : EcoRI+PstI digest of Hin+HPP-A<sub>0</sub>, Lane 4 : unrelated digest, Lane 5 : EcoRI+PstI digest of Hin+HPP-A<sub>1</sub>, Lane 6 : unrelated digest, Lane 7 : EcoRI+PstI digest of Hin+HPP-A<sub>2</sub>]]<br />
<br />
<br />
<br><br />
[[Image:Linebreak.png]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Conclusions==<br />
We were able to successfully insert a 13 amino acid ''hixC'' site into Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), maintaining the functionality of the proteins and allowing for flipping of the DNA by Hin Recombinase. When both split RFP and split GFP are present in the same cell, a yellow color can be observed by eye under UV light, and both red and green fluorescence are detected by fluorimetry. We thus attempted to construct a simple bacterial computer capable of solving a 3-node Hamiltonian Path Problem. We planned to select for the solution by picking cells that fluoresced yellow (both red and green).<br />
<br />
To test for flipping of our HPP computer constructs, we attatched a Hin-expression vector to the HPP construct and observed phenotypic differences in colonies. For all of the starting orientations, we observed colonies of varying colors, suggesting that some colonies had flipped away from their starting orientation and into a new orientation that produced a different phenotype. We also tested for flipping by PCR and were able to see multiple bands for all of our flipped constructs. These results strongly suggest that flipping has occured in each of our HPP constructs. <br />
<br />
In the coming days, we plan to retransform our flipped HPP plasmid (without Hin-Recombinase) into T7 RNAP cells in order to obtain clonal colonies. We hope that selecting for a yellow colony and sequencing this plasmid would give us the solution to the Hamiltonian Path Problem that was programmed into the cells. <br />
<br />
<br><br />
<hr><br />
<center><br />
<[[Davidson Missouri W/Gene splitting | Previous Section]] | [[Davidson Missouri W/Traveling Salesperson Problem | Next Section>]]<br />
</center></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_W/ResultsDavidson Missouri W/Results2007-10-27T01:17:37Z<p>Macampbell: /* Flipping Detection by Phenotype */</p>
<hr />
<div><center>[[Davidson Missouri W| <span style="color:red">Home</span>]] | [[Davidson Missouri W/Background Information| <span style="color:red">Background Information</span>]] | [[Davidson Missouri W/Solving the HPP in vivo| <span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]] | [[Davidson Missouri W/Mathematical Modeling| <span style="color:red">Mathematical Modeling</span>]] | [[Davidson Missouri W/Gene splitting| <span style="color:red">Gene Splitting</span>]] | [[Davidson Missouri W/Results| <span style="color:black">Results</span>]] | [[Davidson Missouri W/Traveling Salesperson Problem| <span style="color:red">Traveling Salesperson Problem</span> ]] | [[Davidson Missouri W/Software|<span style="color:red">Software</span>]] | [[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]</center><br />
<br />
<hr><br />
<br><br />
<br />
==Gene Splitting Worked==<br />
We were able to split two genes: GFP and RFP. Split GFP has strong green fluorescence. Split RFP's red color is much reduced compared to wild-type RFP. It takes overnight incubation at room temperature for the red color to be visible in white light. Both colors are fluorescent under UV light, although the green color predominates. In [https://2007.igem.org/Image:GFPplates.jpg Figure 1] below, a negative-control (on the left) does not fluoresce, but split GFP (on the right) does. [https://2007.igem.org/Image:Split_RFP.png Figure 2] shows a plate of cells containing split RFP.<br />
<br />
[[Image:GFPplates.jpg|thumb|left|500px|'''Figure 1''': The negative control (hixC-GFP2) on the left does not fluoresce, but the experimental split GFP (pLac-RBS-GFP1-hixC-GFP2) on the right does.]]<br />
<br />
[[Image:Split_RFP.png|thumb|left|250px|'''Figure 2''': Cells containing split RFP (pLac-RBS-RFP1-hixC-RFP2) after overnight incubation at room temperature.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Graphs==<br />
<br />
Once we managed to split two genes, we proceeded to implement two different 3-node graphs into plasmids. These graphs were named Graphs A and B and are shown below. For each graph, we wanted to find a Hamiltonian Path from the node represented by RFP (colored in green) to the node represented by the transcriptional terminator (colored in red). The plasmid representation of Graph A is not be capable of flipping into a [https://2007.igem.org/Davidson_Missouri_W/Mathematical_Modeling#True_Positives_and_False_Positives false positive] orientation, while the plasmid representation of Graph B does provide that possibility.<br />
<br />
[[Image:MWSUgraph.png|thumb|200px|left|'''Graph A''': This graph contains 3-nodes and 3-edges. We wanted to find a Hamiltonian path from the node representing RFP (colored in green) to the node representing the transcriptional terminator (colored in red).]] [[Image:DCgraph.png|thumb|200px|none|'''Graph B''': This graph also contains 3-nodes and 3-edges, however it provides the possibility of a false positive solution.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Starting Orientations==<br />
We constructed the following Hamiltonian Path Problem (HPP) constructs to test our bacterial computer system for Graphs A and B. Constructs that are built to solve [https://2007.igem.org/Image:MWSUgraph.png Graph A] are labeled HPP-A. Constructs that are built to solve [https://2007.igem.org/Image:DCgraph.png Graph B] are labeled HPP-B. A subscript equal to 0 denotes a plasmid in a solved orientation. A subscript that is greater than 0 denotes a plasmid that is in an unordered starting orientation.<br />
<br />
*'''HPP<sub>0</sub> (Positive Control for HPP-A and HPP-B constructs) '''<br>[[Image:Picture_1.png]]<br><br />
*'''HPP-A<sub>0</sub> (Solved Orientation)'''<br>[[Image:Picture_2.png]]<br><br />
*'''HPP-A<sub>1</sub> '''<br>[[Image:Picture_3.png]]<br><br />
*'''HPP-A<sub>2</sub> '''<br>[[Image:Picture_4.png]]<br><br />
*'''HPP-B<sub>1</sub> '''<br>[[Image:Picture_5.png]]<br><br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Controls==<br />
Because are graphs require that solved colonies be selected for by double fluorescence (both red and green), we first performed control experiments to ensure that we could distinguish solved and unsolved colonies. To test fluorescence phenotypes when both split GFP and split RFP are the same cell, we used our positive control constructs ([http://partsregistry.org/Part:BBa_I715045 HPP<sub>0</sub>] and [http://partsregistry.org/Part:BBa_I715042 HPP-A<sub>0</sub>]) with split RFP and split GFP both downstream of the T7 polymerase promoter. In the presence of T7 polymerase, cells with the split RFP/GFP plasmid should demonstrate both green and red fluorescence. <br />
<br />
We first tried cotransforming our HPP<sub>0</sub> construct with a T7 RNA polymerase plasmid construct ([http://partsregistry.org/Part:BBa_I715038 BBa_I715038]). As can be seen in [https://2007.igem.org/Image:SplitRFP-GFP.jpg Figure 3] below, this cotransformation resulted in bright green colonies that demonstrated minimal red fluorescence. When we measured the fluorescence of the cotransformed control, RFP was not present at detectable levels (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
We then tried transforming our HPP-A<sub>0</sub> control plasmid into cells that already contained T7 polymerase in their chromosome (T7 RNAP cells). As can be seen in [https://2007.igem.org/Image:DSC02737.jpeg Figure 4] below, these colonies displayed the yellowish color we expected. Based on our fluorescence data, (shown [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]), we were able to show that GFP and RFP were present at detectable levels in this control. We hypothesize that the difference in the two controls is due to higher concentrations of T7 RNA polymerase in the T7 RNAP cells than in the cotransformants. We therefore used the T7 RNAP cells for future experiments.<br />
<br />
[[Image:SplitRFP-GFP.jpg|thumb|left|300px|'''Figure 3''': Colonies containing both the T7 RNA polymerase plasmid and the HPP<sub>0</sub> control plasmid demonstrated strong green fluorescence, but minimal red fluorescence.]]<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|380px|'''Figure 4''': T7 RNAP colonies that contain the HPP-A<sub>0</sub> control plasmid demonstrated the expected yellow fluorescence.]]<br />
[[Image:Linebreak.png]]<br />
<br />
In addition to the regular split RFP/GFP controls, we also tested 2 "hybrid" constructs to ensure that they would not fluoresce. GFP and RFP have similar structures and functions. Previous studies have shown that it is possible to modify GFP to display a wide range of color phenotypes. We created the following parts: Plac-RBS-RFP1-hixC-GFP2 ([http://partsregistry.org/Part:BBa_I715036 BBa_I715036]) and Plac-RBS-GFP1-hixC-RFP2 ([http://partsregistry.org/Part:BBa_I715035 BBa_I715035]). A plasmid may, at some point during its flipping process, contain such sequences. We tested to make sure that the similarity of these two proteins did not make them compatible enough to fluoresce. It was found that neither of these parts show any fluorescence or color change (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Dectection of Fluoresence by Spectroscopy==<br />
We used a fluorometer to measure the fluorescence of various HPP constructs. First, we had to determine the excitation and emission wavelengths that would allow us to detect the green and red fluorescence of split genes. Scans were conducted in order to find these wavelengths. The graphs below show scans of excitation wavelengths that produce fluorescence at an emission wavelength of 515 nm , the wavelength we found to be best for green, and 608 nm, the best wavelength for red. We used these scans to pick a peak emission wavelength for further testing. <br />
<br />
For these scans we used the following constructs:<br><br />
'''pLac-RBS-GFP1-''hixC''-GFP2''' - This construct fluoresces green on the UV box.<br><br />
'''HPP-A<sub>0</sub>''' - This construct fluoresces yellow on the UV box.<br><br />
'''HPP-A<sub>1</sub>''' - This construct fluoresces red on the UV box.<br><br />
'''HPP-A<sub>2</sub>''' - This construct shows no fluorescence on the UV box.<br><br />
<br><br />
<br />
[[Image:GFP Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 515 nm (green light)]]<br />
<br />
[[Image:Red Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 608 nm (red light)]]<br />
<br />
<br><br />
On the basis of these scans, we used excitation/emission wavelengths of 450 nm / 515 nm for green and 560 nm / 608 nm for red to obtain the results shown below.<br />
<br><br />
<br />
===<center>Normalized* Fluorimetry Results</center>===<br />
{| border="1" cellpadding="5" cellspacing="0" align="center" width="100%"<br />
|-<br />
! style="color: black; background-color: white;" | Construct<br />
! style="color: black; background-color: white;" | Fluorescent Color on UV Box<br />
! style="color: black; background-color: white;" | Green<br />
! style="color: black; background-color: white;" | Red<br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span><br />
|Red<br />
|7<br />
|<span style="color:red">263</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span>-RBS-<span style="color:green">GFP</span><br />
|Red<br />
|<span style="color:green">144</span><br />
|<span style="color:red">370</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|Green<br />
|<span style="color:green">136</span><br />
|0<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|0<br />
|<span style="color:red">147</span><br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|11<br />
|2<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|None<br />
|13<br />
|2<br />
|-<br />
<br />
|HPP<sub>0</sub>***<br />
|Green<br />
|<span style="color:green">72</span><br />
|18<br />
|-<br />
<br />
|HPP-A<sub>0</sub><br />
|Yellow<br />
|<span style="color:green">340</span><br />
|<span style="color:red">255</span><br />
|-<br />
<br />
|HPP-A<sub>1</sub><br />
|Red<br />
|1<br />
|<span style="color:red">143</span><br />
|-<br />
<br />
|HPP-A<sub>2</sub><br />
|None<br />
|11<br />
|3<br />
|-<br />
<br />
<br />
|HPP-B<sub>1</sub>***<br />
|hybrid green<br />
|15<br />
|3<br />
|-<br />
<br />
|}<br />
'* To normalize the data, 57 fluorescence units were subtracted from the Green column and 18 from the Red column. These were the lowest fluorescence values for each wavelength.<br><br />
'*** These 2 constructs were cotransformed with the T7 RNA polymerase plasmid (as opposed to being transformed into T7 RNAP cells). This could explain their reduced fluorescence.<br />
<br />
<br><br />
<br />
==Solution Detected by Phenotype==<br />
<br />
To test for flipping by phenotype, we used our 3 - [[Davidson_Missouri_W/Results#Constructs |Graph A]] constructs from above, HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>. As expected, unflipped HPP-A<sub>0</sub> fluoresced yellow, unflipped HPP-A<sub>1</sub> fluoresced red, and unflipped HPP-A<sub>2</sub> demonstrated no fluorescence when transformed into the T7 RNAP cells (See Figures 5-7 below).<br />
<br />
A fragment containing the Hin expression cassette (pLac-RBS-Hin-LVA, [http://partsregistry.org/Part:BBa_S03536 BBa_S03536]) was ligated in front of each of the 3 HPP-A constructs. These HPP-A + Hin plasmids were then transformed into T7 RNAP cells. A colony from each transformation was picked and grown overnight for plasmid mini-prep. Each of the 3 constructs was restriction digested and the insert sizes were verified to be correct. The 3 constructs were then retransformed into T7 RNAP cells, and the resulting transformation mixture was streaked for colony isolation. '''The HPP-A<sub>0</sub> + Hin plate contained mostly yellow colonies but also red and green (See Figures 8 and 11). The Hin HPP-A<sub>1</sub> + Hin plate contained red, green and yellow colonies (See Figures 9 and 12). The HPP-A<sub>2</sub> + Hin plate contained mostly nonfluorescent colonies with a few green and red colonies (See Figures 10 and 13).'''<br><br />
<br />
<br />
''Click twice on the images below for higher resolution.''<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|270px|'''Figure 5:''' HPP-A<sub>0</sub> - These colonies fluoresced yellow]] [[Image:DSC02738.jpeg|thumb|left|270px|'''Figure 6:''' HPP-A<sub>1</sub> - These colonies fluoresced red]] [[Image:DSC02739.jpeg|thumb|left|270px|'''Figure 7:''' HPP-A<sub>2</sub> - These colonies do not fluoresce]]<br />
<br />
[[Image:DSC02746.jpeg|thumb|left|270px|'''Figure 8:''' HPP-A<sub>0</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02744.jpeg|thumb|left|270px|'''Figure 9:''' HPP-A<sub>1</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02736.jpeg|thumb|left|270px|'''Figure 10:''' HPP-A<sub>2</sub> + Hin - The addition of Hin has produced green and red colonies]]<br />
<br />
[[Image:Hin-ABC-cropped2.JPG|thumb|left|270px|'''Figure 11:''' HPP-A<sub>0</sub> + Hin - closeup of Figure 8]] <br />
[[Image:Hin-ACB-cropped3.JPG|thumb|left|270px|'''Figure 12:''' HPP-A<sub>1</sub> + Hin - closeup of Figure 9]]<br />
[[Image:Hin-BAC-cropped.JPG|thumb|left|270px|'''Figure 13:''' HPP-A<sub>2</sub> + Hin - closeup of Figure 10]]<br />
[[Image:Linebreak.png]]<br />
[[Image:Hin-ABC-high.jpg|thumb|left|270px|'''Figure 14:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-high.jpg|thumb|left|270px|'''Figure 15:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-high.jpg|thumb|left|270px|'''Figure 16:''' HPP-A<sub>2</sub> + Hin]]<br />
[[Image:Hin-ABC-low.jpg|thumb|left|270px|'''Figure 16:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-low.jpg|thumb|left|270px|'''Figure 17:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-low.jpg|thumb|left|270px|'''Figure 18:''' HPP-A<sub>2</sub> + Hin]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Flipping Detection by PCR==<br />
<br />
In order to provide physical evidence of Hin-mediated DNA rearrangement of our HPP-A + Hin constructs, we designed PCR primers that would allow us to determine the position of the GFP2 gene half. The experiment below shows the results of PCR using the following multiplex primers: the universal reverse primer [http://partsregistry.org/Part:BBa_G00101 G00101], which binds to the 3' biobrick suffix, a forward primer that binds to RFP1, and a GFP2-forward primer. <br><br><br />
Here is how the primers bind to the 3 HPP-A constructs:<br><br />
[[Image:PCR with Primers.jpeg.png|frame|none|400px|'''PCR Primer Binding''': (from top to bottom) HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>]]<br><br />
<br><br />
[[Image:PCR 10-25-07.JPG|thumb|left|270px|Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lanes 2 : HPP-A<sub>0</sub>, Lanes 3 : HPP-A<sub>1</sub>, Lanes 4 : HPP-A<sub>2</sub>, Lanes 5 : Hin+HPP-A<sub>0</sub>, Lanes 6 : Hin+HPP-A<sub>1</sub>, Lanes 7 : Hin+HPP-A<sub>2</sub>]]<br />
<br><br />
[[Image:gels_10_25_07.JPG|thumb|left|270px|'''Left Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lanes 2,3 : ClaI digest of Hin+HPP-A<sub>0</sub>, Lanes 4,5 : ClaI digest of Hin+HPP-A<sub>1</sub>, Lanes 6,7 : ClaI digest of Hin+HPP-A<sub>2</sub><br><br />
'''Right Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lane 2 : unrelated digest, Lane 3 : EcoRI+PstI digest of Hin+HPP-A<sub>0</sub>, Lane 4 : unrelated digest, Lane 5 : EcoRI+PstI digest of Hin+HPP-A<sub>1</sub>, Lane 6 : unrelated digest, Lane 7 : EcoRI+PstI digest of Hin+HPP-A<sub>2</sub>]]<br />
<br />
<br />
<br><br />
[[Image:Linebreak.png]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Conclusions==<br />
We were able to successfully insert a 13 amino acid ''hixC'' site into Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), maintaining the functionality of the proteins and allowing for flipping of the DNA by Hin Recombinase. When both split RFP and split GFP are present in the same cell, a yellow color can be observed by eye under UV light, and both red and green fluorescence are detected by fluorimetry. We thus attempted to construct a simple bacterial computer capable of solving a 3-node Hamiltonian Path Problem. We planned to select for the solution by picking cells that fluoresced yellow (both red and green).<br />
<br />
To test for flipping of our HPP computer constructs, we attatched a Hin-expression vector to the HPP construct and observed phenotypic differences in colonies. For all of the starting orientations, we observed colonies of varying colors, suggesting that some colonies had flipped away from their starting orientation and into a new orientation that produced a different phenotype. We also tested for flipping by PCR and were able to see multiple bands for all of our flipped constructs. These results strongly suggest that flipping has occured in each of our HPP constructs. <br />
<br />
In the coming days, we plan to retransform our flipped HPP plasmid (without Hin-Recombinase) into T7 RNAP cells in order to obtain clonal colonies. We hope that selecting for a yellow colony and sequencing this plasmid would give us the solution to the Hamiltonian Path Problem that was programmed into the cells. <br />
<br />
<br><br />
<hr><br />
<center><br />
<[[Davidson Missouri W/Gene splitting | Previous Section]] | [[Davidson Missouri W/Traveling Salesperson Problem | Next Section>]]<br />
</center></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_W/ResultsDavidson Missouri W/Results2007-10-27T01:16:46Z<p>Macampbell: /* Gene Splitting */</p>
<hr />
<div><center>[[Davidson Missouri W| <span style="color:red">Home</span>]] | [[Davidson Missouri W/Background Information| <span style="color:red">Background Information</span>]] | [[Davidson Missouri W/Solving the HPP in vivo| <span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]] | [[Davidson Missouri W/Mathematical Modeling| <span style="color:red">Mathematical Modeling</span>]] | [[Davidson Missouri W/Gene splitting| <span style="color:red">Gene Splitting</span>]] | [[Davidson Missouri W/Results| <span style="color:black">Results</span>]] | [[Davidson Missouri W/Traveling Salesperson Problem| <span style="color:red">Traveling Salesperson Problem</span> ]] | [[Davidson Missouri W/Software|<span style="color:red">Software</span>]] | [[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]</center><br />
<br />
<hr><br />
<br><br />
<br />
==Gene Splitting Worked==<br />
We were able to split two genes: GFP and RFP. Split GFP has strong green fluorescence. Split RFP's red color is much reduced compared to wild-type RFP. It takes overnight incubation at room temperature for the red color to be visible in white light. Both colors are fluorescent under UV light, although the green color predominates. In [https://2007.igem.org/Image:GFPplates.jpg Figure 1] below, a negative-control (on the left) does not fluoresce, but split GFP (on the right) does. [https://2007.igem.org/Image:Split_RFP.png Figure 2] shows a plate of cells containing split RFP.<br />
<br />
[[Image:GFPplates.jpg|thumb|left|500px|'''Figure 1''': The negative control (hixC-GFP2) on the left does not fluoresce, but the experimental split GFP (pLac-RBS-GFP1-hixC-GFP2) on the right does.]]<br />
<br />
[[Image:Split_RFP.png|thumb|left|250px|'''Figure 2''': Cells containing split RFP (pLac-RBS-RFP1-hixC-RFP2) after overnight incubation at room temperature.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Graphs==<br />
<br />
Once we managed to split two genes, we proceeded to implement two different 3-node graphs into plasmids. These graphs were named Graphs A and B and are shown below. For each graph, we wanted to find a Hamiltonian Path from the node represented by RFP (colored in green) to the node represented by the transcriptional terminator (colored in red). The plasmid representation of Graph A is not be capable of flipping into a [https://2007.igem.org/Davidson_Missouri_W/Mathematical_Modeling#True_Positives_and_False_Positives false positive] orientation, while the plasmid representation of Graph B does provide that possibility.<br />
<br />
[[Image:MWSUgraph.png|thumb|200px|left|'''Graph A''': This graph contains 3-nodes and 3-edges. We wanted to find a Hamiltonian path from the node representing RFP (colored in green) to the node representing the transcriptional terminator (colored in red).]] [[Image:DCgraph.png|thumb|200px|none|'''Graph B''': This graph also contains 3-nodes and 3-edges, however it provides the possibility of a false positive solution.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Starting Orientations==<br />
We constructed the following Hamiltonian Path Problem (HPP) constructs to test our bacterial computer system for Graphs A and B. Constructs that are built to solve [https://2007.igem.org/Image:MWSUgraph.png Graph A] are labeled HPP-A. Constructs that are built to solve [https://2007.igem.org/Image:DCgraph.png Graph B] are labeled HPP-B. A subscript equal to 0 denotes a plasmid in a solved orientation. A subscript that is greater than 0 denotes a plasmid that is in an unordered starting orientation.<br />
<br />
*'''HPP<sub>0</sub> (Positive Control for HPP-A and HPP-B constructs) '''<br>[[Image:Picture_1.png]]<br><br />
*'''HPP-A<sub>0</sub> (Solved Orientation)'''<br>[[Image:Picture_2.png]]<br><br />
*'''HPP-A<sub>1</sub> '''<br>[[Image:Picture_3.png]]<br><br />
*'''HPP-A<sub>2</sub> '''<br>[[Image:Picture_4.png]]<br><br />
*'''HPP-B<sub>1</sub> '''<br>[[Image:Picture_5.png]]<br><br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Controls==<br />
Because are graphs require that solved colonies be selected for by double fluorescence (both red and green), we first performed control experiments to ensure that we could distinguish solved and unsolved colonies. To test fluorescence phenotypes when both split GFP and split RFP are the same cell, we used our positive control constructs ([http://partsregistry.org/Part:BBa_I715045 HPP<sub>0</sub>] and [http://partsregistry.org/Part:BBa_I715042 HPP-A<sub>0</sub>]) with split RFP and split GFP both downstream of the T7 polymerase promoter. In the presence of T7 polymerase, cells with the split RFP/GFP plasmid should demonstrate both green and red fluorescence. <br />
<br />
We first tried cotransforming our HPP<sub>0</sub> construct with a T7 RNA polymerase plasmid construct ([http://partsregistry.org/Part:BBa_I715038 BBa_I715038]). As can be seen in [https://2007.igem.org/Image:SplitRFP-GFP.jpg Figure 3] below, this cotransformation resulted in bright green colonies that demonstrated minimal red fluorescence. When we measured the fluorescence of the cotransformed control, RFP was not present at detectable levels (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
We then tried transforming our HPP-A<sub>0</sub> control plasmid into cells that already contained T7 polymerase in their chromosome (T7 RNAP cells). As can be seen in [https://2007.igem.org/Image:DSC02737.jpeg Figure 4] below, these colonies displayed the yellowish color we expected. Based on our fluorescence data, (shown [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]), we were able to show that GFP and RFP were present at detectable levels in this control. We hypothesize that the difference in the two controls is due to higher concentrations of T7 RNA polymerase in the T7 RNAP cells than in the cotransformants. We therefore used the T7 RNAP cells for future experiments.<br />
<br />
[[Image:SplitRFP-GFP.jpg|thumb|left|300px|'''Figure 3''': Colonies containing both the T7 RNA polymerase plasmid and the HPP<sub>0</sub> control plasmid demonstrated strong green fluorescence, but minimal red fluorescence.]]<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|380px|'''Figure 4''': T7 RNAP colonies that contain the HPP-A<sub>0</sub> control plasmid demonstrated the expected yellow fluorescence.]]<br />
[[Image:Linebreak.png]]<br />
<br />
In addition to the regular split RFP/GFP controls, we also tested 2 "hybrid" constructs to ensure that they would not fluoresce. GFP and RFP have similar structures and functions. Previous studies have shown that it is possible to modify GFP to display a wide range of color phenotypes. We created the following parts: Plac-RBS-RFP1-hixC-GFP2 ([http://partsregistry.org/Part:BBa_I715036 BBa_I715036]) and Plac-RBS-GFP1-hixC-RFP2 ([http://partsregistry.org/Part:BBa_I715035 BBa_I715035]). A plasmid may, at some point during its flipping process, contain such sequences. We tested to make sure that the similarity of these two proteins did not make them compatible enough to fluoresce. It was found that neither of these parts show any fluorescence or color change (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Dectection of Fluoresence by Spectroscopy==<br />
We used a fluorometer to measure the fluorescence of various HPP constructs. First, we had to determine the excitation and emission wavelengths that would allow us to detect the green and red fluorescence of split genes. Scans were conducted in order to find these wavelengths. The graphs below show scans of excitation wavelengths that produce fluorescence at an emission wavelength of 515 nm , the wavelength we found to be best for green, and 608 nm, the best wavelength for red. We used these scans to pick a peak emission wavelength for further testing. <br />
<br />
For these scans we used the following constructs:<br><br />
'''pLac-RBS-GFP1-''hixC''-GFP2''' - This construct fluoresces green on the UV box.<br><br />
'''HPP-A<sub>0</sub>''' - This construct fluoresces yellow on the UV box.<br><br />
'''HPP-A<sub>1</sub>''' - This construct fluoresces red on the UV box.<br><br />
'''HPP-A<sub>2</sub>''' - This construct shows no fluorescence on the UV box.<br><br />
<br><br />
<br />
[[Image:GFP Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 515 nm (green light)]]<br />
<br />
[[Image:Red Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 608 nm (red light)]]<br />
<br />
<br><br />
On the basis of these scans, we used excitation/emission wavelengths of 450 nm / 515 nm for green and 560 nm / 608 nm for red to obtain the results shown below.<br />
<br><br />
<br />
===<center>Normalized* Fluorimetry Results</center>===<br />
{| border="1" cellpadding="5" cellspacing="0" align="center" width="100%"<br />
|-<br />
! style="color: black; background-color: white;" | Construct<br />
! style="color: black; background-color: white;" | Fluorescent Color on UV Box<br />
! style="color: black; background-color: white;" | Green<br />
! style="color: black; background-color: white;" | Red<br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span><br />
|Red<br />
|7<br />
|<span style="color:red">263</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span>-RBS-<span style="color:green">GFP</span><br />
|Red<br />
|<span style="color:green">144</span><br />
|<span style="color:red">370</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|Green<br />
|<span style="color:green">136</span><br />
|0<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|0<br />
|<span style="color:red">147</span><br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|11<br />
|2<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|None<br />
|13<br />
|2<br />
|-<br />
<br />
|HPP<sub>0</sub>***<br />
|Green<br />
|<span style="color:green">72</span><br />
|18<br />
|-<br />
<br />
|HPP-A<sub>0</sub><br />
|Yellow<br />
|<span style="color:green">340</span><br />
|<span style="color:red">255</span><br />
|-<br />
<br />
|HPP-A<sub>1</sub><br />
|Red<br />
|1<br />
|<span style="color:red">143</span><br />
|-<br />
<br />
|HPP-A<sub>2</sub><br />
|None<br />
|11<br />
|3<br />
|-<br />
<br />
<br />
|HPP-B<sub>1</sub>***<br />
|hybrid green<br />
|15<br />
|3<br />
|-<br />
<br />
|}<br />
'* To normalize the data, 57 fluorescence units were subtracted from the Green column and 18 from the Red column. These were the lowest fluorescence values for each wavelength.<br><br />
'*** These 2 constructs were cotransformed with the T7 RNA polymerase plasmid (as opposed to being transformed into T7 RNAP cells). This could explain their reduced fluorescence.<br />
<br />
<br><br />
<br />
==Flipping Detection by Phenotype==<br />
<br />
To test for flipping by phenotype, we used our 3 - [[Davidson_Missouri_W/Results#Constructs |Graph A]] constructs from above, HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>. As expected, unflipped HPP-A<sub>0</sub> fluoresced yellow, unflipped HPP-A<sub>1</sub> fluoresced red, and unflipped HPP-A<sub>2</sub> demonstrated no fluorescence when transformed into the T7 RNAP cells (See Figures 5-7 below).<br />
<br />
A fragment containing the Hin expression cassette (pLac-RBS-Hin-LVA, [http://partsregistry.org/Part:BBa_S03536 BBa_S03536]) was ligated in front of each of the 3 HPP-A constructs. These HPP-A + Hin plasmids were then transformed into T7 RNAP cells. A colony from each transformation was picked and grown overnight for plasmid mini-prep. Each of the 3 constructs was restriction digested and the insert sizes were verified to be correct. The 3 constructs were then retransformed into T7 RNAP cells, and the resulting transformation mixture was streaked for colony isolation. '''The HPP-A<sub>0</sub> + Hin plate contained mostly yellow colonies but also red and green (See Figures 8 and 11). The Hin HPP-A<sub>1</sub> + Hin plate contained red, green and yellow colonies (See Figures 9 and 12). The HPP-A<sub>2</sub> + Hin plate contained mostly nonfluorescent colonies with a few green and red colonies (See Figures 10 and 13).'''<br><br />
<br />
<br />
''Click twice on the images below for higher resolution.''<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|270px|'''Figure 5:''' HPP-A<sub>0</sub> - These colonies fluoresced yellow]] [[Image:DSC02738.jpeg|thumb|left|270px|'''Figure 6:''' HPP-A<sub>1</sub> - These colonies fluoresced red]] [[Image:DSC02739.jpeg|thumb|left|270px|'''Figure 7:''' HPP-A<sub>2</sub> - These colonies do not fluoresce]]<br />
<br />
[[Image:DSC02746.jpeg|thumb|left|270px|'''Figure 8:''' HPP-A<sub>0</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02744.jpeg|thumb|left|270px|'''Figure 9:''' HPP-A<sub>1</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02736.jpeg|thumb|left|270px|'''Figure 10:''' HPP-A<sub>2</sub> + Hin - The addition of Hin has produced green and red colonies]]<br />
<br />
[[Image:Hin-ABC-cropped2.JPG|thumb|left|270px|'''Figure 11:''' HPP-A<sub>0</sub> + Hin - closeup of Figure 8]] <br />
[[Image:Hin-ACB-cropped3.JPG|thumb|left|270px|'''Figure 12:''' HPP-A<sub>1</sub> + Hin - closeup of Figure 9]]<br />
[[Image:Hin-BAC-cropped.JPG|thumb|left|270px|'''Figure 13:''' HPP-A<sub>2</sub> + Hin - closeup of Figure 10]]<br />
[[Image:Linebreak.png]]<br />
[[Image:Hin-ABC-high.jpg|thumb|left|270px|'''Figure 14:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-high.jpg|thumb|left|270px|'''Figure 15:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-high.jpg|thumb|left|270px|'''Figure 16:''' HPP-A<sub>2</sub> + Hin]]<br />
[[Image:Hin-ABC-low.jpg|thumb|left|270px|'''Figure 16:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-low.jpg|thumb|left|270px|'''Figure 17:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-low.jpg|thumb|left|270px|'''Figure 18:''' HPP-A<sub>2</sub> + Hin]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Flipping Detection by PCR==<br />
<br />
In order to provide physical evidence of Hin-mediated DNA rearrangement of our HPP-A + Hin constructs, we designed PCR primers that would allow us to determine the position of the GFP2 gene half. The experiment below shows the results of PCR using the following multiplex primers: the universal reverse primer [http://partsregistry.org/Part:BBa_G00101 G00101], which binds to the 3' biobrick suffix, a forward primer that binds to RFP1, and a GFP2-forward primer. <br><br><br />
Here is how the primers bind to the 3 HPP-A constructs:<br><br />
[[Image:PCR with Primers.jpeg.png|frame|none|400px|'''PCR Primer Binding''': (from top to bottom) HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>]]<br><br />
<br><br />
[[Image:PCR 10-25-07.JPG|thumb|left|270px|Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lanes 2 : HPP-A<sub>0</sub>, Lanes 3 : HPP-A<sub>1</sub>, Lanes 4 : HPP-A<sub>2</sub>, Lanes 5 : Hin+HPP-A<sub>0</sub>, Lanes 6 : Hin+HPP-A<sub>1</sub>, Lanes 7 : Hin+HPP-A<sub>2</sub>]]<br />
<br><br />
[[Image:gels_10_25_07.JPG|thumb|left|270px|'''Left Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lanes 2,3 : ClaI digest of Hin+HPP-A<sub>0</sub>, Lanes 4,5 : ClaI digest of Hin+HPP-A<sub>1</sub>, Lanes 6,7 : ClaI digest of Hin+HPP-A<sub>2</sub><br><br />
'''Right Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lane 2 : unrelated digest, Lane 3 : EcoRI+PstI digest of Hin+HPP-A<sub>0</sub>, Lane 4 : unrelated digest, Lane 5 : EcoRI+PstI digest of Hin+HPP-A<sub>1</sub>, Lane 6 : unrelated digest, Lane 7 : EcoRI+PstI digest of Hin+HPP-A<sub>2</sub>]]<br />
<br />
<br />
<br><br />
[[Image:Linebreak.png]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Conclusions==<br />
We were able to successfully insert a 13 amino acid ''hixC'' site into Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), maintaining the functionality of the proteins and allowing for flipping of the DNA by Hin Recombinase. When both split RFP and split GFP are present in the same cell, a yellow color can be observed by eye under UV light, and both red and green fluorescence are detected by fluorimetry. We thus attempted to construct a simple bacterial computer capable of solving a 3-node Hamiltonian Path Problem. We planned to select for the solution by picking cells that fluoresced yellow (both red and green).<br />
<br />
To test for flipping of our HPP computer constructs, we attatched a Hin-expression vector to the HPP construct and observed phenotypic differences in colonies. For all of the starting orientations, we observed colonies of varying colors, suggesting that some colonies had flipped away from their starting orientation and into a new orientation that produced a different phenotype. We also tested for flipping by PCR and were able to see multiple bands for all of our flipped constructs. These results strongly suggest that flipping has occured in each of our HPP constructs. <br />
<br />
In the coming days, we plan to retransform our flipped HPP plasmid (without Hin-Recombinase) into T7 RNAP cells in order to obtain clonal colonies. We hope that selecting for a yellow colony and sequencing this plasmid would give us the solution to the Hamiltonian Path Problem that was programmed into the cells. <br />
<br />
<br><br />
<hr><br />
<center><br />
<[[Davidson Missouri W/Gene splitting | Previous Section]] | [[Davidson Missouri W/Traveling Salesperson Problem | Next Section>]]<br />
</center></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-10-27T01:16:07Z<p>Macampbell: /* Our Successful Project */</p>
<hr />
<div><center>[[Davidson Missouri W| <span style="color:black">Home</span>]] | [[Davidson Missouri W/Background Information| <span style="color:red">Background Information</span>]] | [[Davidson Missouri W/Solving the HPP in vivo| <span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]] | [[Davidson Missouri W/Mathematical Modeling| <span style="color:red">Mathematical Modeling</span>]] | [[Davidson Missouri W/Gene splitting| <span style="color:red">Gene Splitting</span>]] | [[Davidson Missouri W/Results| <span style="color:red">Results</span>]] | [[Davidson Missouri W/Traveling Salesperson Problem| <span style="color:red">Traveling Salesperson Problem</span> ]] | [[Davidson Missouri W/Software|<span style="color:red">Software</span>]] | [[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]</center><br />
<br />
<hr><br />
<br><br />
[[Image:dmw_logo2.png|center]]<br />
<br />
[[Image:Computer.png|center]]<br />
<br />
<center><br />
=The Team=<br />
</center><br />
{| border="1" cellpadding="5" cellspacing="0" align="center" width="100%"<br />
|-<br />
! style="color: white; background-color: black;"| The Team<br />
! style="color: white; background-color: black;" | The Faculty<br />
! style="color: white; background-color: black;" | Team Logos<br />
! style="color: white; background-color: black;" | Group Photo<br />
|-<br />
<br />
|style="color: black; background-color: red;" align="center"| '''Davidson'''<br />
<b><br />
[[Davidson Missouri W/Oyinade Adefuye|<span style="color:black">Oyinade Adefuye</span>]]<br />
<br><br />
[[Davidson Missouri W/Will DeLoache|<span style="color:black">Will DeLoache</span>]]<br />
<br><br />
[[Davidson Missouri W/Jim Dickson|<span style="color:black">Jim Dickson</span>]]<br />
<br><br />
[[Davidson Missouri W/Andrew Martens|<span style="color:black">Andrew Martens</span>]]<br />
<br><br />
[[Davidson Missouri W/Amber Shoecraft|<span style="color:black">Amber Shoecraft</span>]]<br />
<br><br />
[[Davidson Missouri W/Mike Waters|<span style="color:black">Mike Waters</span>]]<br />
</b><br />
<br />
|style="color: black; background-color: red;" align="center"|<br />
<b><br />
[[Davidson Missouri W/A. Malcolm Campbell|<span style="color:black">A. Malcolm Campbell</span>]]<br />
<br><br />
[[Davidson Missouri W/Karmella Haynes|<span style="color:black">Karmella Haynes</span>]]<br />
<br><br />
[[Davidson Missouri W/Laurie Heyer|<span style="color:black">Laurie Heyer</span>]]<br />
</b><br />
<br />
|style="color: black; background-color: white;" align="center"|<br />
[[Image:DavidsonLogo.gif]]<br />
<br />
|style="color: black; background-color: white;" align="center"|<br />
[[Image:Team1.jpg|thumb|center|300px]]<br />
<br />
|-<br />
<br />
|style="color: black; background-color: gold;" align="center"|'''Missouri Western'''<br />
<b><br />
[[Davidson Missouri W/Jordan Baumgardner|<span style="color:black;">Jordan Baumgardner</span>]]<br />
<br><br />
[[Davidson Missouri W/Tom Crowley|<span style="color:black;">Tom Crowley</span>]]<br />
<br><br />
[[Davidson Missouri W/Lane H. Heard|<span style="color:black;">Lane H. Heard</span>]]<br />
<br><br />
[[Davidson Missouri W/Nickolaus Morton|<span style="color:black;">Nickolaus Morton</span>]]<br />
<br><br />
[[Davidson Missouri W/Michelle Ritter|<span style="color:black;">Michelle Ritter</span>]]<br />
<br><br />
[[Davidson Missouri W/Jessica Treece|<span style="color:black;">Jessica Treece</span>]]<br />
<br><br />
[[Davidson Missouri W/Matthew Unzicker|<span style="color:black;">Matthew Unzicker</span>]]<br />
<br><br />
[[Davidson Missouri W/Amanda Valencia|<span style="color:black;">Amanda Valencia</span>]]<br />
</b><br />
<br />
|style="color: black; background-color: gold;" align="center"|<br />
<b><br />
[[Davidson Missouri W/Todd Eckdahl|<span style="color:black;">Todd Eckdahl</span>]]<br />
<br><br />
[[Davidson Missouri W/Jeff Poet|<span style="color:black;">Jeff Poet</span>]]<br />
</b><br />
<br />
|style="color: black; background-color: white;" align="center"|[[Image:MWLogo.gif]]<br />
<br />
|style="color: black; background-color: white;" align="center"|[[Image:MWSUteam.jpeg|thumb|center|300px]]<br />
|-<br />
<br />
|}<br />
<br><br />
<center><br />
<br />
=Our Successful Project=<br />
</center><br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center" width="90%"<br />
|-<br />
! style="color: black; background-color: red;" width="20%"| <font size="+1">In Depth</font><br />
! colspan="3" style="color: black; background-color: red;" width="60%"| <font size="+1">Overview</font><br />
|-<br />
|style="color: black; background-color: black;" align="center"|<br />
[[Davidson Missouri W/Background Information|<span style="color:red">Background Information</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Solving the HPP in vivo|<span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Mathematical Modeling|<span style="color:red">Mathematical Modeling</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Gene splitting|<span style="color:red">Gene Splitting</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Results|<span style="color:red">Results</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Traveling Salesperson Problem|<span style="color:red">Traveling Salesperson Problem</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Software|<span style="color:red">Software</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]<br />
<br><br><Br><br />
|Hamiltonian Path Problem<br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/''hix'' DNA recombination mechanism which exists in nature in ''Salmonella'' as standard biobricks for use in ''E. coli''. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we successfully continued our collaboration and our efforts to manipulate ''E. coli'' into mathematics problem solvers as we refine our efforts with the hin/''hix'' mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adleman in 1994 (see [[Davidson_Missouri_W/Resources_and_Citations | citations]]) where a unique Hamiltonian path was found ''in vitro'' for a particular directed graph on seven nodes. We were able to use bacterial computers to solve the Hamiltonian path problem ''in vivo''. ([[Davidson Missouri W/Background Information#Why Use Bacteria?|Why use a bacterial computer?]])<br />
<br />
<br> <br />
<br />
[[Image:Adelman.png|thumb|300px|center|The Adleman graph.]] <br />
<br />
<center> For the graph used in Adleman's paper (shown above), the Hamiltonian Path Problem would ask: can you find a path along the directed edges that travels from node 1 (green) to node 5 (red) and visits each node on the graph exactly once? <br><br />
[https://static.igem.org/mediawiki/2007/6/6f/Adelmansolution.png Click here] for the solution.<br />
</center><br />
|}<br />
<br />
<br><br />
<center> '''A Human Representation of the Adleman Graph.'''<br />
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</center></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-10-27T01:13:54Z<p>Macampbell: /* Our Project */</p>
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<div><center>[[Davidson Missouri W| <span style="color:black">Home</span>]] | [[Davidson Missouri W/Background Information| <span style="color:red">Background Information</span>]] | [[Davidson Missouri W/Solving the HPP in vivo| <span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]] | [[Davidson Missouri W/Mathematical Modeling| <span style="color:red">Mathematical Modeling</span>]] | [[Davidson Missouri W/Gene splitting| <span style="color:red">Gene Splitting</span>]] | [[Davidson Missouri W/Results| <span style="color:red">Results</span>]] | [[Davidson Missouri W/Traveling Salesperson Problem| <span style="color:red">Traveling Salesperson Problem</span> ]] | [[Davidson Missouri W/Software|<span style="color:red">Software</span>]] | [[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]</center><br />
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{| border="1" cellpadding="5" cellspacing="0" align="center" width="100%"<br />
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|style="color: black; background-color: red;" align="center"| '''Davidson'''<br />
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[[Davidson Missouri W/Oyinade Adefuye|<span style="color:black">Oyinade Adefuye</span>]]<br />
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[[Davidson Missouri W/Will DeLoache|<span style="color:black">Will DeLoache</span>]]<br />
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[[Davidson Missouri W/Jim Dickson|<span style="color:black">Jim Dickson</span>]]<br />
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[[Davidson Missouri W/Andrew Martens|<span style="color:black">Andrew Martens</span>]]<br />
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[[Davidson Missouri W/Amber Shoecraft|<span style="color:black">Amber Shoecraft</span>]]<br />
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[[Davidson Missouri W/Mike Waters|<span style="color:black">Mike Waters</span>]]<br />
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|style="color: black; background-color: red;" align="center"|<br />
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[[Davidson Missouri W/A. Malcolm Campbell|<span style="color:black">A. Malcolm Campbell</span>]]<br />
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[[Davidson Missouri W/Karmella Haynes|<span style="color:black">Karmella Haynes</span>]]<br />
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[[Davidson Missouri W/Laurie Heyer|<span style="color:black">Laurie Heyer</span>]]<br />
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|style="color: black; background-color: gold;" align="center"|'''Missouri Western'''<br />
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[[Davidson Missouri W/Tom Crowley|<span style="color:black;">Tom Crowley</span>]]<br />
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[[Davidson Missouri W/Lane H. Heard|<span style="color:black;">Lane H. Heard</span>]]<br />
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[[Davidson Missouri W/Nickolaus Morton|<span style="color:black;">Nickolaus Morton</span>]]<br />
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[[Davidson Missouri W/Michelle Ritter|<span style="color:black;">Michelle Ritter</span>]]<br />
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[[Davidson Missouri W/Jessica Treece|<span style="color:black;">Jessica Treece</span>]]<br />
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[[Davidson Missouri W/Matthew Unzicker|<span style="color:black;">Matthew Unzicker</span>]]<br />
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[[Davidson Missouri W/Amanda Valencia|<span style="color:black;">Amanda Valencia</span>]]<br />
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[[Davidson Missouri W/Jeff Poet|<span style="color:black;">Jeff Poet</span>]]<br />
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{| border="1" cellpadding="5" cellspacing="0" align="center" width="90%"<br />
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[[Davidson Missouri W/Background Information|<span style="color:red">Background Information</span>]]<br />
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[[Davidson Missouri W/Solving the HPP in vivo|<span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]]<br />
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[[Davidson Missouri W/Mathematical Modeling|<span style="color:red">Mathematical Modeling</span>]]<br />
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[[Davidson Missouri W/Traveling Salesperson Problem|<span style="color:red">Traveling Salesperson Problem</span>]]<br />
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[[Davidson Missouri W/Software|<span style="color:red">Software</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]<br />
<br><br><Br><br />
|Hamiltonian Path Problem<br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/''hix'' DNA recombination mechanism which exists in nature in ''Salmonella'' as standard biobricks for use in ''E. coli''. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we successfully continued our collaboration and our efforts to manipulate ''E. coli'' into mathematics problem solvers as we refine our efforts with the hin/''hix'' mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adleman in 1994 (see [[Davidson_Missouri_W/Resources_and_Citations | citations]]) where a unique Hamiltonian path was found ''in vitro'' for a particular directed graph on seven nodes. We propose to make progress toward solving the particular problem ''in vivo''. ([[Davidson Missouri W/Background Information#Why Use Bacteria?|Why use a bacterial computer?]])<br />
<br />
<br> <br />
<br />
[[Image:Adelman.png|thumb|300px|center|The Adleman graph.]] <br />
<br />
<center> For the graph used in Adleman's paper (shown above), the Hamiltonian Path Problem would ask: can you find a path along the directed edges that travels from node 1 (green) to node 5 (red) and visits each node on the graph exactly once? <br><br />
[https://static.igem.org/mediawiki/2007/6/6f/Adelmansolution.png Click here] for the solution.<br />
</center><br />
|}<br />
<br />
<br><br />
<center> '''A Human Representation of the Adleman Graph.'''<br />
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</center></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-10-27T01:13:19Z<p>Macampbell: /* Our Project */</p>
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<div><center>[[Davidson Missouri W| <span style="color:black">Home</span>]] | [[Davidson Missouri W/Background Information| <span style="color:red">Background Information</span>]] | [[Davidson Missouri W/Solving the HPP in vivo| <span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]] | [[Davidson Missouri W/Mathematical Modeling| <span style="color:red">Mathematical Modeling</span>]] | [[Davidson Missouri W/Gene splitting| <span style="color:red">Gene Splitting</span>]] | [[Davidson Missouri W/Results| <span style="color:red">Results</span>]] | [[Davidson Missouri W/Traveling Salesperson Problem| <span style="color:red">Traveling Salesperson Problem</span> ]] | [[Davidson Missouri W/Software|<span style="color:red">Software</span>]] | [[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]</center><br />
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{| border="1" cellpadding="5" cellspacing="0" align="center" width="100%"<br />
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|style="color: black; background-color: red;" align="center"| '''Davidson'''<br />
<b><br />
[[Davidson Missouri W/Oyinade Adefuye|<span style="color:black">Oyinade Adefuye</span>]]<br />
<br><br />
[[Davidson Missouri W/Will DeLoache|<span style="color:black">Will DeLoache</span>]]<br />
<br><br />
[[Davidson Missouri W/Jim Dickson|<span style="color:black">Jim Dickson</span>]]<br />
<br><br />
[[Davidson Missouri W/Andrew Martens|<span style="color:black">Andrew Martens</span>]]<br />
<br><br />
[[Davidson Missouri W/Amber Shoecraft|<span style="color:black">Amber Shoecraft</span>]]<br />
<br><br />
[[Davidson Missouri W/Mike Waters|<span style="color:black">Mike Waters</span>]]<br />
</b><br />
<br />
|style="color: black; background-color: red;" align="center"|<br />
<b><br />
[[Davidson Missouri W/A. Malcolm Campbell|<span style="color:black">A. Malcolm Campbell</span>]]<br />
<br><br />
[[Davidson Missouri W/Karmella Haynes|<span style="color:black">Karmella Haynes</span>]]<br />
<br><br />
[[Davidson Missouri W/Laurie Heyer|<span style="color:black">Laurie Heyer</span>]]<br />
</b><br />
<br />
|style="color: black; background-color: white;" align="center"|<br />
[[Image:DavidsonLogo.gif]]<br />
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|style="color: black; background-color: white;" align="center"|<br />
[[Image:Team1.jpg|thumb|center|300px]]<br />
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|-<br />
<br />
|style="color: black; background-color: gold;" align="center"|'''Missouri Western'''<br />
<b><br />
[[Davidson Missouri W/Jordan Baumgardner|<span style="color:black;">Jordan Baumgardner</span>]]<br />
<br><br />
[[Davidson Missouri W/Tom Crowley|<span style="color:black;">Tom Crowley</span>]]<br />
<br><br />
[[Davidson Missouri W/Lane H. Heard|<span style="color:black;">Lane H. Heard</span>]]<br />
<br><br />
[[Davidson Missouri W/Nickolaus Morton|<span style="color:black;">Nickolaus Morton</span>]]<br />
<br><br />
[[Davidson Missouri W/Michelle Ritter|<span style="color:black;">Michelle Ritter</span>]]<br />
<br><br />
[[Davidson Missouri W/Jessica Treece|<span style="color:black;">Jessica Treece</span>]]<br />
<br><br />
[[Davidson Missouri W/Matthew Unzicker|<span style="color:black;">Matthew Unzicker</span>]]<br />
<br><br />
[[Davidson Missouri W/Amanda Valencia|<span style="color:black;">Amanda Valencia</span>]]<br />
</b><br />
<br />
|style="color: black; background-color: gold;" align="center"|<br />
<b><br />
[[Davidson Missouri W/Todd Eckdahl|<span style="color:black;">Todd Eckdahl</span>]]<br />
<br><br />
[[Davidson Missouri W/Jeff Poet|<span style="color:black;">Jeff Poet</span>]]<br />
</b><br />
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|style="color: black; background-color: white;" align="center"|[[Image:MWLogo.gif]]<br />
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|style="color: black; background-color: white;" align="center"|[[Image:MWSUteam.jpeg|thumb|center|300px]]<br />
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<center><br />
<br />
=Our Project=<br />
</center><br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center" width="90%"<br />
|-<br />
! style="color: black; background-color: red;" width="20%"| <font size="+1">In Depth</font><br />
! colspan="3" style="color: black; background-color: red;" width="60%"| <font size="+1">Overview</font><br />
|-<br />
|style="color: black; background-color: black;" align="center"|<br />
[[Davidson Missouri W/Background Information|<span style="color:red">Background Information</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Solving the HPP in vivo|<span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Mathematical Modeling|<span style="color:red">Mathematical Modeling</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Gene splitting|<span style="color:red">Gene Splitting</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Results|<span style="color:red">Results</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Traveling Salesperson Problem|<span style="color:red">Traveling Salesperson Problem</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Software|<span style="color:red">Software</span>]]<br />
<br><br><br><br />
[[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]<br />
<br><br><Br><br />
|Hamiltonian Path Problem<br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/''hix'' DNA recombination mechanism which exists in nature in ''Salmonella'' as standard biobricks for use in ''E. coli''. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we successfully continued our collaboration and our efforts to manipulate ''E. coli'' into mathematics problem solvers as we refine our efforts with the hin/''hix'' mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adleman in 1994 (see [[Davidson_Missouri_W/Resources_and_Citations | citations]]) where a unique Hamiltonian path was found ''in vitro'' for a particular directed graph on seven nodes. We propose to make progress toward solving the particular problem ''in vivo''. ([[Davidson Missouri W/Background Information#Why Use Bacteria?|Why use a bacterial computer?]])<br />
<br />
<br> <br />
<br />
[[Image:Adelman.png|thumb|300px|center|The Adleman graph.]] <br />
<br />
<center> For the graph used in Adleman's paper (shown above), the Hamiltonian Path Problem would ask: can you find a path along the directed edges that travels from node 1 (green) to node 5 (red) and visits each node on the graph exactly once? <br><br />
[https://static.igem.org/mediawiki/2007/6/6f/Adelmansolution.png Click here] for the solution.<br />
</center><br />
|}<br />
<br />
<br><br />
<center> '''A Human Representation of the Adleman Graph.'''<br />
<br />
<html><br />
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<Previous Section | [[Davidson Missouri W/Background Information | Next Section>]]<br />
</center></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_W/ResultsDavidson Missouri W/Results2007-10-26T21:16:22Z<p>Macampbell: /* <center>Normalized* Fluorimetry Results</center> */</p>
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<div><center>[[Davidson Missouri W| <span style="color:red">Home</span>]] | [[Davidson Missouri W/Background Information| <span style="color:red">Background Information</span>]] | [[Davidson Missouri W/Solving the HPP in vivo| <span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]] | [[Davidson Missouri W/Mathematical Modeling| <span style="color:red">Mathematical Modeling</span>]] | [[Davidson Missouri W/Gene splitting| <span style="color:red">Gene Splitting</span>]] | [[Davidson Missouri W/Results| <span style="color:black">Results</span>]] | [[Davidson Missouri W/Traveling Salesperson Problem| <span style="color:red">Traveling Salesperson Problem</span> ]] | [[Davidson Missouri W/Software|<span style="color:red">Software</span>]] | [[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]</center><br />
<br />
<hr><br />
<br><br />
<br />
==Gene Splitting==<br />
We were able to split two genes: GFP and RFP. Split GFP has strong green fluorescence. Split RFP's red color is much reduced compared to wild-type RFP. It takes overnight incubation at room temperature for the red color to be visible in white light. Both colors are fluorescent under UV light, although the green color predominates. In [https://2007.igem.org/Image:GFPplates.jpg Figure 1] below, a negative-control (on the left) does not fluoresce, but split GFP (on the right) does. [https://2007.igem.org/Image:Split_RFP.png Figure 2] shows a plate of cells containing split RFP.<br />
<br />
[[Image:GFPplates.jpg|thumb|left|500px|'''Figure 1''': The negative control (hixC-GFP2) on the left does not fluoresce, but the experimental split GFP (pLac-RBS-GFP1-hixC-GFP2) on the right does.]]<br />
<br />
[[Image:Split_RFP.png|thumb|left|250px|'''Figure 2''': Cells containing split RFP (pLac-RBS-RFP1-hixC-RFP2) after overnight incubation at room temperature.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Graphs==<br />
<br />
Once we managed to split two genes, we proceeded to implement two different 3-node graphs into plasmids. These graphs were named Graphs A and B and are shown below. For each graph, we wanted to find a Hamiltonian Path from the node represented by RFP (colored in green) to the node represented by the transcriptional terminator (colored in red). The plasmid representation of Graph A is not be capable of flipping into a [https://2007.igem.org/Davidson_Missouri_W/Mathematical_Modeling#True_Positives_and_False_Positives false positive] orientation, while the plasmid representation of Graph B does provide that possibility.<br />
<br />
[[Image:MWSUgraph.png|thumb|200px|left|'''Graph A''': This graph contains 3-nodes and 3-edges. We wanted to find a Hamiltonian path from the node representing RFP (colored in green) to the node representing the transcriptional terminator (colored in red).]] [[Image:DCgraph.png|thumb|200px|none|'''Graph B''': This graph also contains 3-nodes and 3-edges, however it provides the possibility of a false positive solution.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Starting Orientations==<br />
We constructed the following Hamiltonian Path Problem (HPP) constructs to test our bacterial computer system for Graphs A and B. Constructs that are built to solve [https://2007.igem.org/Image:MWSUgraph.png Graph A] are labeled HPP-A. Constructs that are built to solve [https://2007.igem.org/Image:DCgraph.png Graph B] are labeled HPP-B. A subscript equal to 0 denotes a plasmid in a solved orientation. A subscript that is greater than 0 denotes a plasmid that is in an unordered starting orientation.<br />
<br />
*'''HPP<sub>0</sub> (Positive Control for HPP-A and HPP-B constructs) '''<br>[[Image:Picture_1.png]]<br><br />
*'''HPP-A<sub>0</sub> (Solved Orientation)'''<br>[[Image:Picture_2.png]]<br><br />
*'''HPP-A<sub>1</sub> '''<br>[[Image:Picture_3.png]]<br><br />
*'''HPP-A<sub>2</sub> '''<br>[[Image:Picture_4.png]]<br><br />
*'''HPP-B<sub>1</sub> '''<br>[[Image:Picture_5.png]]<br><br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Controls==<br />
Because are graphs require that solved colonies be selected for by double fluorescence (both red and green), we first performed control experiments to ensure that we could distinguish solved and unsolved colonies. To test fluorescence phenotypes when both split GFP and split RFP are the same cell, we used our positive control constructs ([http://partsregistry.org/Part:BBa_I715045 HPP<sub>0</sub>] and [http://partsregistry.org/Part:BBa_I715042 HPP-A<sub>0</sub>]) with split RFP and split GFP both downstream of the T7 polymerase promoter. In the presence of T7 polymerase, cells with the split RFP/GFP plasmid should demonstrate both green and red fluorescence. <br />
<br />
We first tried cotransforming our HPP<sub>0</sub> construct with a T7 RNA polymerase plasmid construct ([http://partsregistry.org/Part:BBa_I715038 BBa_I715038]). As can be seen in [https://2007.igem.org/Image:SplitRFP-GFP.jpg Figure 3] below, this cotransformation resulted in bright green colonies that demonstrated minimal red fluorescence. When we measured the fluorescence of the cotransformed control, RFP was not present at detectable levels (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
We then tried transforming our HPP-A<sub>0</sub> control plasmid into cells that already contained T7 polymerase in their chromosome (T7 RNAP cells). As can be seen in [https://2007.igem.org/Image:DSC02737.jpeg Figure 4] below, these colonies displayed the yellowish color we expected. Based on our fluorescence data, (shown [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]), we were able to show that GFP and RFP were present at detectable levels in this control. We hypothesize that the difference in the two controls is due to higher concentrations of T7 RNA polymerase in the T7 RNAP cells than in the cotransformants. We therefore used the T7 RNAP cells for future experiments.<br />
<br />
[[Image:SplitRFP-GFP.jpg|thumb|left|300px|'''Figure 3''': Colonies containing both the T7 RNA polymerase plasmid and the HPP<sub>0</sub> control plasmid demonstrated strong green fluorescence, but minimal red fluorescence.]]<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|380px|'''Figure 4''': T7 RNAP colonies that contain the HPP-A<sub>0</sub> control plasmid demonstrated the expected yellow fluorescence.]]<br />
[[Image:Linebreak.png]]<br />
<br />
In addition to the regular split RFP/GFP controls, we also tested 2 "hybrid" constructs to ensure that they would not fluoresce. GFP and RFP have similar structures and functions. Previous studies have shown that it is possible to modify GFP to display a wide range of color phenotypes. We created the following parts: Plac-RBS-RFP1-hixC-GFP2 ([http://partsregistry.org/Part:BBa_I715036 BBa_I715036]) and Plac-RBS-GFP1-hixC-RFP2 ([http://partsregistry.org/Part:BBa_I715035 BBa_I715035]). A plasmid may, at some point during its flipping process, contain such sequences. We tested to make sure that the similarity of these two proteins did not make them compatible enough to fluoresce. It was found that neither of these parts show any fluorescence or color change (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Dectection of Fluoresence by Spectroscopy==<br />
We used a fluorometer to measure the fluorescence of various HPP constructs. First, we had to determine the excitation and emission wavelengths that would allow us to detect the green and red fluorescence of split genes. Scans were conducted in order to find these wavelengths. The graphs below show scans of excitation wavelengths that produce fluorescence at an emission wavelength of 515 nm , the wavelength we found to be best for green, and 608 nm, the best wavelength for red. We used these scans to pick a peak emission wavelength for further testing. <br />
<br />
For these scans we used the following constructs:<br><br />
'''pLac-RBS-GFP1-''hixC''-GFP2''' - This construct fluoresces green on the UV box.<br><br />
'''HPP-A<sub>0</sub>''' - This construct fluoresces yellow on the UV box.<br><br />
'''HPP-A<sub>1</sub>''' - This construct fluoresces red on the UV box.<br><br />
'''HPP-A<sub>2</sub>''' - This construct shows no fluorescence on the UV box.<br><br />
<br><br />
<br />
[[Image:GFP Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 515 nm (green light)]]<br />
<br />
[[Image:Red Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 608 nm (red light)]]<br />
<br />
<br><br />
On the basis of these scans, we used excitation/emission wavelengths of 450 nm / 515 nm for green and 560 nm / 608 nm for red to obtain the results shown below.<br />
<br><br />
<br />
===<center>Normalized* Fluorimetry Results</center>===<br />
{| border="1" cellpadding="5" cellspacing="0" align="center" width="100%"<br />
|-<br />
! style="color: black; background-color: white;" | Construct<br />
! style="color: black; background-color: white;" | Fluorescent Color on UV Box<br />
! style="color: black; background-color: white;" | Green<br />
! style="color: black; background-color: white;" | Red<br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span><br />
|Red<br />
|7<br />
|<span style="color:red">263</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span>-RBS-<span style="color:green">GFP</span><br />
|Red<br />
|<span style="color:green">144</span><br />
|<span style="color:red">370</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|Green<br />
|<span style="color:green">136</span><br />
|0<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|0<br />
|<span style="color:red">147</span><br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|11<br />
|2<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|None<br />
|13<br />
|2<br />
|-<br />
<br />
|HPP<sub>0</sub>***<br />
|Green<br />
|<span style="color:green">72</span><br />
|18<br />
|-<br />
<br />
|HPP-A<sub>0</sub><br />
|Yellow<br />
|<span style="color:green">340</span><br />
|<span style="color:red">255</span><br />
|-<br />
<br />
|HPP-A<sub>1</sub><br />
|Red<br />
|1<br />
|<span style="color:red">143</span><br />
|-<br />
<br />
|HPP-A<sub>2</sub><br />
|None<br />
|11<br />
|3<br />
|-<br />
<br />
<br />
|HPP-B<sub>1</sub>***<br />
|hybrid green<br />
|15<br />
|3<br />
|-<br />
<br />
|}<br />
'* To normalize the data, 57 fluorescence units were subtracted from the Green column and 18 from the Red column. These were the lowest fluorescence values for each wavelength.<br><br />
'*** These 2 constructs were cotransformed with the T7 RNA polymerase plasmid (as opposed to being transformed into T7 RNAP cells). This could explain their reduced fluorescence.<br />
<br />
<br><br />
<br />
==Flipping Detection by Phenotype==<br />
<br />
To test for flipping by phenotype, we used our 3 - [[Davidson_Missouri_W/Results#Constructs |Graph A]] constructs from above, HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>. As expected, unflipped HPP-A<sub>0</sub> fluoresced yellow, unflipped HPP-A<sub>1</sub> fluoresced red, and unflipped HPP-A<sub>2</sub> demonstrated no fluorescence when transformed into the T7 RNAP cells (See Figures 5-7 below).<br />
<br />
A fragment containing the Hin expression cassette (pLac-RBS-Hin-LVA, [http://partsregistry.org/Part:BBa_S03536 BBa_S03536]) was ligated in front of each of the 3 HPP-A constructs. These HPP-A + Hin plasmids were then transformed into T7 RNAP cells. A colony from each transformation was picked and grown overnight for plasmid mini-prep. Each of the 3 constructs was restriction digested and the insert sizes were verified to be correct. The 3 constructs were then retransformed into T7 RNAP cells, and the resulting transformation mixture was streaked for colony isolation. '''The HPP-A<sub>0</sub> + Hin plate contained mostly yellow colonies but also red and green (See Figures 8 and 11). The Hin HPP-A<sub>1</sub> + Hin plate contained red, green and yellow colonies (See Figures 9 and 12). The HPP-A<sub>2</sub> + Hin plate contained mostly nonfluorescent colonies with a few green and red colonies (See Figures 10 and 13).'''<br><br />
<br />
<br />
''Click twice on the images below for higher resolution.''<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|270px|'''Figure 5:''' HPP-A<sub>0</sub> - These colonies fluoresced yellow]] [[Image:DSC02738.jpeg|thumb|left|270px|'''Figure 6:''' HPP-A<sub>1</sub> - These colonies fluoresced red]] [[Image:DSC02739.jpeg|thumb|left|270px|'''Figure 7:''' HPP-A<sub>2</sub> - These colonies do not fluoresce]]<br />
<br />
[[Image:DSC02746.jpeg|thumb|left|270px|'''Figure 8:''' HPP-A<sub>0</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02744.jpeg|thumb|left|270px|'''Figure 9:''' HPP-A<sub>1</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02736.jpeg|thumb|left|270px|'''Figure 10:''' HPP-A<sub>2</sub> + Hin - The addition of Hin has produced green and red colonies]]<br />
<br />
[[Image:Hin-ABC-cropped2.JPG|thumb|left|270px|'''Figure 11:''' HPP-A<sub>0</sub> + Hin - closeup of Figure 8]] <br />
[[Image:Hin-ACB-cropped3.JPG|thumb|left|270px|'''Figure 12:''' HPP-A<sub>1</sub> + Hin - closeup of Figure 9]]<br />
[[Image:Hin-BAC-cropped.JPG|thumb|left|270px|'''Figure 13:''' HPP-A<sub>2</sub> + Hin - closeup of Figure 10]]<br />
[[Image:Linebreak.png]]<br />
[[Image:Hin-ABC-high.jpg|thumb|left|270px|'''Figure 14:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-high.jpg|thumb|left|270px|'''Figure 15:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-high.jpg|thumb|left|270px|'''Figure 16:''' HPP-A<sub>2</sub> + Hin]]<br />
[[Image:Hin-ABC-low.jpg|thumb|left|270px|'''Figure 16:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-low.jpg|thumb|left|270px|'''Figure 17:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-low.jpg|thumb|left|270px|'''Figure 18:''' HPP-A<sub>2</sub> + Hin]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Flipping Detection by PCR==<br />
<br />
In order to provide physical evidence of Hin-mediated DNA rearrangement of our HPP-A + Hin constructs, we designed PCR primers that would allow us to determine the position of the GFP2 gene half. The experiment below shows the results of PCR using the universal reverse primer [http://partsregistry.org/Part:BBa_G00101 G00101], which binds to the 3' biobrick suffix, and a GFP2-forward primer. <br><br><br />
Here is how the primers bind to the 3 HPP-A constructs:<br><br />
[[Image:HPP-A_PCR_Primers.png|frame|none|400px|'''PCR Primer Binding''': (from top to bottom) HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>]]<br><br />
<br><br />
[[Image:PCR 10-25-07.JPG|thumb|left|270px|Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lanes 2 : HPP-A<sub>0</sub>, Lanes 3 : HPP-A<sub>1</sub>, Lanes 4 : HPP-A<sub>2</sub>, Lanes 5 : Hin+HPP-A<sub>0</sub>, Lanes 6 : Hin+HPP-A<sub>1</sub>, Lanes 7 : Hin+HPP-A<sub>2</sub>]]<br />
<br><br />
[[Image:gels_10_25_07.JPG|thumb|left|270px|'''Left Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lanes 2,3 : ClaI digest of Hin+HPP-A<sub>0</sub>, Lanes 4,5 : ClaI digest of Hin+HPP-A<sub>1</sub>, Lanes 6,7 : ClaI digest of Hin+HPP-A<sub>2</sub><br><br />
'''Right Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lane 2 : unrelated digest, Lane 3 : EcoRI+PstI digest of Hin+HPP-A<sub>0</sub>, Lane 4 : unrelated digest, Lane 5 : EcoRI+PstI digest of Hin+HPP-A<sub>1</sub>, Lane 6 : unrelated digest, Lane 7 : EcoRI+PstI digest of Hin+HPP-A<sub>2</sub>]]<br />
<br />
<br />
<br><br />
[[Image:Linebreak.png]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Conclusions==<br />
We were able to successfully insert a 13 amino acid ''hixC'' site into Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), maintaining the functionality of the proteins and allowing for flipping of the DNA by Hin Recombinase. When both split RFP and split GFP are present in the same cell, a yellow color can be observed by eye under UV light, and both red and green fluorescence are detected by fluorimetry. We thus attempted to construct a simple bacterial computer capable of solving a 3-node Hamiltonian Path Problem. We planned to select for the solution by picking cells that fluoresced yellow (both red and green).<br />
<br />
To test for flipping of our HPP computer constructs, we attatched a Hin-expression vector to the HPP construct and observed phenotypic differences in colonies. For all of the starting orientations, we observed colonies of varying colors, suggesting that some colonies had flipped away from their starting orientation and into a new orientation that produced a different phenotype. We also tested for flipping by PCR and were able to see multiple bands for all of our flipped constructs. These results strongly suggest that flipping has occured in each of our HPP constructs. <br />
<br />
In the coming days, we plan to retransform our flipped HPP plasmid (without Hin-Recombinase) into T7 RNAP cells in order to obtain clonal colonies. We hope that selecting for a yellow colony and sequencing this plasmid would give us the solution to the Hamiltonian Path Problem that was programmed into the cells. <br />
<br />
<br><br />
<hr><br />
<center><br />
<[[Davidson Missouri W/Gene splitting | Previous Section]] | [[Davidson Missouri W/Traveling Salesperson Problem | Next Section>]]<br />
</center></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_W/ResultsDavidson Missouri W/Results2007-10-26T21:15:56Z<p>Macampbell: /* <center>Normalized* Fluorimetry Results</center> */</p>
<hr />
<div><center>[[Davidson Missouri W| <span style="color:red">Home</span>]] | [[Davidson Missouri W/Background Information| <span style="color:red">Background Information</span>]] | [[Davidson Missouri W/Solving the HPP in vivo| <span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]] | [[Davidson Missouri W/Mathematical Modeling| <span style="color:red">Mathematical Modeling</span>]] | [[Davidson Missouri W/Gene splitting| <span style="color:red">Gene Splitting</span>]] | [[Davidson Missouri W/Results| <span style="color:black">Results</span>]] | [[Davidson Missouri W/Traveling Salesperson Problem| <span style="color:red">Traveling Salesperson Problem</span> ]] | [[Davidson Missouri W/Software|<span style="color:red">Software</span>]] | [[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]</center><br />
<br />
<hr><br />
<br><br />
<br />
==Gene Splitting==<br />
We were able to split two genes: GFP and RFP. Split GFP has strong green fluorescence. Split RFP's red color is much reduced compared to wild-type RFP. It takes overnight incubation at room temperature for the red color to be visible in white light. Both colors are fluorescent under UV light, although the green color predominates. In [https://2007.igem.org/Image:GFPplates.jpg Figure 1] below, a negative-control (on the left) does not fluoresce, but split GFP (on the right) does. [https://2007.igem.org/Image:Split_RFP.png Figure 2] shows a plate of cells containing split RFP.<br />
<br />
[[Image:GFPplates.jpg|thumb|left|500px|'''Figure 1''': The negative control (hixC-GFP2) on the left does not fluoresce, but the experimental split GFP (pLac-RBS-GFP1-hixC-GFP2) on the right does.]]<br />
<br />
[[Image:Split_RFP.png|thumb|left|250px|'''Figure 2''': Cells containing split RFP (pLac-RBS-RFP1-hixC-RFP2) after overnight incubation at room temperature.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Graphs==<br />
<br />
Once we managed to split two genes, we proceeded to implement two different 3-node graphs into plasmids. These graphs were named Graphs A and B and are shown below. For each graph, we wanted to find a Hamiltonian Path from the node represented by RFP (colored in green) to the node represented by the transcriptional terminator (colored in red). The plasmid representation of Graph A is not be capable of flipping into a [https://2007.igem.org/Davidson_Missouri_W/Mathematical_Modeling#True_Positives_and_False_Positives false positive] orientation, while the plasmid representation of Graph B does provide that possibility.<br />
<br />
[[Image:MWSUgraph.png|thumb|200px|left|'''Graph A''': This graph contains 3-nodes and 3-edges. We wanted to find a Hamiltonian path from the node representing RFP (colored in green) to the node representing the transcriptional terminator (colored in red).]] [[Image:DCgraph.png|thumb|200px|none|'''Graph B''': This graph also contains 3-nodes and 3-edges, however it provides the possibility of a false positive solution.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Starting Orientations==<br />
We constructed the following Hamiltonian Path Problem (HPP) constructs to test our bacterial computer system for Graphs A and B. Constructs that are built to solve [https://2007.igem.org/Image:MWSUgraph.png Graph A] are labeled HPP-A. Constructs that are built to solve [https://2007.igem.org/Image:DCgraph.png Graph B] are labeled HPP-B. A subscript equal to 0 denotes a plasmid in a solved orientation. A subscript that is greater than 0 denotes a plasmid that is in an unordered starting orientation.<br />
<br />
*'''HPP<sub>0</sub> (Positive Control for HPP-A and HPP-B constructs) '''<br>[[Image:Picture_1.png]]<br><br />
*'''HPP-A<sub>0</sub> (Solved Orientation)'''<br>[[Image:Picture_2.png]]<br><br />
*'''HPP-A<sub>1</sub> '''<br>[[Image:Picture_3.png]]<br><br />
*'''HPP-A<sub>2</sub> '''<br>[[Image:Picture_4.png]]<br><br />
*'''HPP-B<sub>1</sub> '''<br>[[Image:Picture_5.png]]<br><br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Controls==<br />
Because are graphs require that solved colonies be selected for by double fluorescence (both red and green), we first performed control experiments to ensure that we could distinguish solved and unsolved colonies. To test fluorescence phenotypes when both split GFP and split RFP are the same cell, we used our positive control constructs ([http://partsregistry.org/Part:BBa_I715045 HPP<sub>0</sub>] and [http://partsregistry.org/Part:BBa_I715042 HPP-A<sub>0</sub>]) with split RFP and split GFP both downstream of the T7 polymerase promoter. In the presence of T7 polymerase, cells with the split RFP/GFP plasmid should demonstrate both green and red fluorescence. <br />
<br />
We first tried cotransforming our HPP<sub>0</sub> construct with a T7 RNA polymerase plasmid construct ([http://partsregistry.org/Part:BBa_I715038 BBa_I715038]). As can be seen in [https://2007.igem.org/Image:SplitRFP-GFP.jpg Figure 3] below, this cotransformation resulted in bright green colonies that demonstrated minimal red fluorescence. When we measured the fluorescence of the cotransformed control, RFP was not present at detectable levels (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
We then tried transforming our HPP-A<sub>0</sub> control plasmid into cells that already contained T7 polymerase in their chromosome (T7 RNAP cells). As can be seen in [https://2007.igem.org/Image:DSC02737.jpeg Figure 4] below, these colonies displayed the yellowish color we expected. Based on our fluorescence data, (shown [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]), we were able to show that GFP and RFP were present at detectable levels in this control. We hypothesize that the difference in the two controls is due to higher concentrations of T7 RNA polymerase in the T7 RNAP cells than in the cotransformants. We therefore used the T7 RNAP cells for future experiments.<br />
<br />
[[Image:SplitRFP-GFP.jpg|thumb|left|300px|'''Figure 3''': Colonies containing both the T7 RNA polymerase plasmid and the HPP<sub>0</sub> control plasmid demonstrated strong green fluorescence, but minimal red fluorescence.]]<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|380px|'''Figure 4''': T7 RNAP colonies that contain the HPP-A<sub>0</sub> control plasmid demonstrated the expected yellow fluorescence.]]<br />
[[Image:Linebreak.png]]<br />
<br />
In addition to the regular split RFP/GFP controls, we also tested 2 "hybrid" constructs to ensure that they would not fluoresce. GFP and RFP have similar structures and functions. Previous studies have shown that it is possible to modify GFP to display a wide range of color phenotypes. We created the following parts: Plac-RBS-RFP1-hixC-GFP2 ([http://partsregistry.org/Part:BBa_I715036 BBa_I715036]) and Plac-RBS-GFP1-hixC-RFP2 ([http://partsregistry.org/Part:BBa_I715035 BBa_I715035]). A plasmid may, at some point during its flipping process, contain such sequences. We tested to make sure that the similarity of these two proteins did not make them compatible enough to fluoresce. It was found that neither of these parts show any fluorescence or color change (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Dectection of Fluoresence by Spectroscopy==<br />
We used a fluorometer to measure the fluorescence of various HPP constructs. First, we had to determine the excitation and emission wavelengths that would allow us to detect the green and red fluorescence of split genes. Scans were conducted in order to find these wavelengths. The graphs below show scans of excitation wavelengths that produce fluorescence at an emission wavelength of 515 nm , the wavelength we found to be best for green, and 608 nm, the best wavelength for red. We used these scans to pick a peak emission wavelength for further testing. <br />
<br />
For these scans we used the following constructs:<br><br />
'''pLac-RBS-GFP1-''hixC''-GFP2''' - This construct fluoresces green on the UV box.<br><br />
'''HPP-A<sub>0</sub>''' - This construct fluoresces yellow on the UV box.<br><br />
'''HPP-A<sub>1</sub>''' - This construct fluoresces red on the UV box.<br><br />
'''HPP-A<sub>2</sub>''' - This construct shows no fluorescence on the UV box.<br><br />
<br><br />
<br />
[[Image:GFP Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 515 nm (green light)]]<br />
<br />
[[Image:Red Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 608 nm (red light)]]<br />
<br />
<br><br />
On the basis of these scans, we used excitation/emission wavelengths of 450 nm / 515 nm for green and 560 nm / 608 nm for red to obtain the results shown below.<br />
<br><br />
<br />
===<center>Normalized* Fluorimetry Results</center>===<br />
{| border="1" cellpadding="5" cellspacing="0" align="center" width="100%"<br />
|-<br />
! style="color: black; background-color: white;" | Construct<br />
! style="color: black; background-color: white;" | Fluorescent Color on UV Box<br />
! style="color: black; background-color: white;" | Green<br />
! style="color: black; background-color: white;" | Red<br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span><br />
|Red<br />
|7<br />
|<span style="color:red">263</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span>-RBS-<span style="color:green">GFP</span><br />
|Red<br />
|<span style="color:green">144</span><br />
|<span style="color:red">370</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|Green<br />
|<span style="color:green">136</span><br />
|0<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|0<br />
|<span style="color:red">147</span><br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|11<br />
|2<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|None<br />
|13<br />
|2<br />
|-<br />
<br />
|HPP<sub>0</sub>***<br />
|Green<br />
|<span style="color:green">72</span><br />
|18<br />
|-<br />
<br />
|HPP-A<sub>0</sub><br />
|Yellow<br />
|<span style="color:green">340</span><br />
|<span style="color:red">255</span><br />
|-<br />
<br />
|HPP-A<sub>1</sub><br />
|Red<br />
|1<br />
|<span style="color:red">143</span><br />
|-<br />
<br />
|HPP-A<sub>2</sub><br />
|None<br />
|11<br />
|3<br />
|-<br />
<br />
<br />
|HPP-B<sub>1</sub>***<br />
|hybrid green<br />
|15<br />
|3<br />
|-<br />
<br />
|}<br />
'* To normalize the data, 57 fluorescence units were subtracted from the Green column and 18 from the Red column. These were the lowest fluorescence values for each wavelength.<br><br />
'** We hypothesize that the scan of this construct was faulty, as the cells fluoresce bright green under the UV lamp.<br><br />
'*** These 2 constructs were cotransformed with the T7 RNA polymerase plasmid (as opposed to being transformed into T7 RNAP cells). This could explain their reduced fluorescence.<br />
<br />
<br><br />
<br />
==Flipping Detection by Phenotype==<br />
<br />
To test for flipping by phenotype, we used our 3 - [[Davidson_Missouri_W/Results#Constructs |Graph A]] constructs from above, HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>. As expected, unflipped HPP-A<sub>0</sub> fluoresced yellow, unflipped HPP-A<sub>1</sub> fluoresced red, and unflipped HPP-A<sub>2</sub> demonstrated no fluorescence when transformed into the T7 RNAP cells (See Figures 5-7 below).<br />
<br />
A fragment containing the Hin expression cassette (pLac-RBS-Hin-LVA, [http://partsregistry.org/Part:BBa_S03536 BBa_S03536]) was ligated in front of each of the 3 HPP-A constructs. These HPP-A + Hin plasmids were then transformed into T7 RNAP cells. A colony from each transformation was picked and grown overnight for plasmid mini-prep. Each of the 3 constructs was restriction digested and the insert sizes were verified to be correct. The 3 constructs were then retransformed into T7 RNAP cells, and the resulting transformation mixture was streaked for colony isolation. '''The HPP-A<sub>0</sub> + Hin plate contained mostly yellow colonies but also red and green (See Figures 8 and 11). The Hin HPP-A<sub>1</sub> + Hin plate contained red, green and yellow colonies (See Figures 9 and 12). The HPP-A<sub>2</sub> + Hin plate contained mostly nonfluorescent colonies with a few green and red colonies (See Figures 10 and 13).'''<br><br />
<br />
<br />
''Click twice on the images below for higher resolution.''<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|270px|'''Figure 5:''' HPP-A<sub>0</sub> - These colonies fluoresced yellow]] [[Image:DSC02738.jpeg|thumb|left|270px|'''Figure 6:''' HPP-A<sub>1</sub> - These colonies fluoresced red]] [[Image:DSC02739.jpeg|thumb|left|270px|'''Figure 7:''' HPP-A<sub>2</sub> - These colonies do not fluoresce]]<br />
<br />
[[Image:DSC02746.jpeg|thumb|left|270px|'''Figure 8:''' HPP-A<sub>0</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02744.jpeg|thumb|left|270px|'''Figure 9:''' HPP-A<sub>1</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02736.jpeg|thumb|left|270px|'''Figure 10:''' HPP-A<sub>2</sub> + Hin - The addition of Hin has produced green and red colonies]]<br />
<br />
[[Image:Hin-ABC-cropped2.JPG|thumb|left|270px|'''Figure 11:''' HPP-A<sub>0</sub> + Hin - closeup of Figure 8]] <br />
[[Image:Hin-ACB-cropped3.JPG|thumb|left|270px|'''Figure 12:''' HPP-A<sub>1</sub> + Hin - closeup of Figure 9]]<br />
[[Image:Hin-BAC-cropped.JPG|thumb|left|270px|'''Figure 13:''' HPP-A<sub>2</sub> + Hin - closeup of Figure 10]]<br />
[[Image:Linebreak.png]]<br />
[[Image:Hin-ABC-high.jpg|thumb|left|270px|'''Figure 14:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-high.jpg|thumb|left|270px|'''Figure 15:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-high.jpg|thumb|left|270px|'''Figure 16:''' HPP-A<sub>2</sub> + Hin]]<br />
[[Image:Hin-ABC-low.jpg|thumb|left|270px|'''Figure 16:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-low.jpg|thumb|left|270px|'''Figure 17:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-low.jpg|thumb|left|270px|'''Figure 18:''' HPP-A<sub>2</sub> + Hin]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Flipping Detection by PCR==<br />
<br />
In order to provide physical evidence of Hin-mediated DNA rearrangement of our HPP-A + Hin constructs, we designed PCR primers that would allow us to determine the position of the GFP2 gene half. The experiment below shows the results of PCR using the universal reverse primer [http://partsregistry.org/Part:BBa_G00101 G00101], which binds to the 3' biobrick suffix, and a GFP2-forward primer. <br><br><br />
Here is how the primers bind to the 3 HPP-A constructs:<br><br />
[[Image:HPP-A_PCR_Primers.png|frame|none|400px|'''PCR Primer Binding''': (from top to bottom) HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>]]<br><br />
<br><br />
[[Image:PCR 10-25-07.JPG|thumb|left|270px|Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lanes 2 : HPP-A<sub>0</sub>, Lanes 3 : HPP-A<sub>1</sub>, Lanes 4 : HPP-A<sub>2</sub>, Lanes 5 : Hin+HPP-A<sub>0</sub>, Lanes 6 : Hin+HPP-A<sub>1</sub>, Lanes 7 : Hin+HPP-A<sub>2</sub>]]<br />
<br><br />
[[Image:gels_10_25_07.JPG|thumb|left|270px|'''Left Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lanes 2,3 : ClaI digest of Hin+HPP-A<sub>0</sub>, Lanes 4,5 : ClaI digest of Hin+HPP-A<sub>1</sub>, Lanes 6,7 : ClaI digest of Hin+HPP-A<sub>2</sub><br><br />
'''Right Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000, 6000 bp), Lane 2 : unrelated digest, Lane 3 : EcoRI+PstI digest of Hin+HPP-A<sub>0</sub>, Lane 4 : unrelated digest, Lane 5 : EcoRI+PstI digest of Hin+HPP-A<sub>1</sub>, Lane 6 : unrelated digest, Lane 7 : EcoRI+PstI digest of Hin+HPP-A<sub>2</sub>]]<br />
<br />
<br />
<br><br />
[[Image:Linebreak.png]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Conclusions==<br />
We were able to successfully insert a 13 amino acid ''hixC'' site into Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), maintaining the functionality of the proteins and allowing for flipping of the DNA by Hin Recombinase. When both split RFP and split GFP are present in the same cell, a yellow color can be observed by eye under UV light, and both red and green fluorescence are detected by fluorimetry. We thus attempted to construct a simple bacterial computer capable of solving a 3-node Hamiltonian Path Problem. We planned to select for the solution by picking cells that fluoresced yellow (both red and green).<br />
<br />
To test for flipping of our HPP computer constructs, we attatched a Hin-expression vector to the HPP construct and observed phenotypic differences in colonies. For all of the starting orientations, we observed colonies of varying colors, suggesting that some colonies had flipped away from their starting orientation and into a new orientation that produced a different phenotype. We also tested for flipping by PCR and were able to see multiple bands for all of our flipped constructs. These results strongly suggest that flipping has occured in each of our HPP constructs. <br />
<br />
In the coming days, we plan to retransform our flipped HPP plasmid (without Hin-Recombinase) into T7 RNAP cells in order to obtain clonal colonies. We hope that selecting for a yellow colony and sequencing this plasmid would give us the solution to the Hamiltonian Path Problem that was programmed into the cells. <br />
<br />
<br><br />
<hr><br />
<center><br />
<[[Davidson Missouri W/Gene splitting | Previous Section]] | [[Davidson Missouri W/Traveling Salesperson Problem | Next Section>]]<br />
</center></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_W/ResultsDavidson Missouri W/Results2007-10-26T18:05:16Z<p>Macampbell: /* <center>Normalized* Fluorimetry Results</center> */</p>
<hr />
<div><center>[[Davidson Missouri W| <span style="color:red">Home</span>]] | [[Davidson Missouri W/Background Information| <span style="color:red">Background Information</span>]] | [[Davidson Missouri W/Solving the HPP in vivo| <span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]] | [[Davidson Missouri W/Mathematical Modeling| <span style="color:red">Mathematical Modeling</span>]] | [[Davidson Missouri W/Gene splitting| <span style="color:red">Gene Splitting</span>]] | [[Davidson Missouri W/Results| <span style="color:black">Results</span>]] | [[Davidson Missouri W/Traveling Salesperson Problem| <span style="color:red">Traveling Salesperson Problem</span> ]] | [[Davidson Missouri W/Software|<span style="color:red">Software</span>]] | [[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]</center><br />
<br />
<hr><br />
<br><br />
<br />
==Gene Splitting==<br />
We were able to split two genes: GFP and RFP. Split GFP has strong green fluorescence. Split RFP's red color is much reduced compared to wild-type RFP. It takes overnight incubation at room temperature for the red color to be visible in white light. Both colors are fluorescent under UV light, although the green color predominates. In [https://2007.igem.org/Image:GFPplates.jpg Figure 1] below, a negative-control (on the left) does not fluoresce, but split GFP (on the right) does. [https://2007.igem.org/Image:Split_RFP.png Figure 2] shows a plate of cells containing split RFP.<br />
<br />
[[Image:GFPplates.jpg|thumb|left|500px|'''Figure 1''': The negative control (hixC-GFP2) on the left does not fluoresce, but the experimental split GFP (pLac-RBS-GFP1-hixC-GFP2) on the right does.]]<br />
<br />
[[Image:Split_RFP.png|thumb|left|250px|'''Figure 2''': Cells containing split RFP (pLac-RBS-RFP1-hixC-RFP2) after overnight incubation at room temperature.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Graphs==<br />
<br />
Once we managed to split two genes, we proceeded to implement two different 3-node graphs into plasmids. These graphs were named Graphs A and B and are shown below. For each graph, we wanted to find a Hamiltonian Path from the node represented by RFP (colored in green) to the node represented by the transcriptional terminator (colored in red). The plasmid representation of Graph A is not be capable of flipping into a [https://2007.igem.org/Davidson_Missouri_W/Mathematical_Modeling#True_Positives_and_False_Positives false positive] orientation, while the plasmid representation of Graph B does provide that possibility.<br />
<br />
[[Image:MWSUgraph.png|thumb|200px|left|'''Graph A''': This graph contains 3-nodes and 3-edges. We wanted to find a Hamiltonian path from the node representing RFP (colored in green) to the node representing the transcriptional terminator (colored in red).]] [[Image:DCgraph.png|thumb|200px|none|'''Graph B''': This graph also contains 3-nodes and 3-edges, however it provides the possibility of a false positive solution.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Starting Orientations==<br />
We constructed the following Hamiltonian Path Problem (HPP) constructs to test our bacterial computer system for Graphs A and B. Constructs that are built to solve [https://2007.igem.org/Image:MWSUgraph.png Graph A] are labeled HPP-A. Constructs that are built to solve [https://2007.igem.org/Image:DCgraph.png Graph B] are labeled HPP-B. A subscript equal to 0 denotes a plasmid in a solved orientation. A subscript that is greater than 0 denotes a plasmid that is in an unordered starting orientation.<br />
<br />
*'''HPP<sub>0</sub> (Positive Control for HPP-A and HPP-B constructs) '''<br>[[Image:Picture_1.png]]<br><br />
*'''HPP-A<sub>0</sub> (Solved Orientation)'''<br>[[Image:Picture_2.png]]<br><br />
*'''HPP-A<sub>1</sub> '''<br>[[Image:Picture_3.png]]<br><br />
*'''HPP-A<sub>2</sub> '''<br>[[Image:Picture_4.png]]<br><br />
*'''HPP-B<sub>1</sub> '''<br>[[Image:Picture_5.png]]<br><br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Controls==<br />
Because are graphs require that solved colonies be selected for by double fluorescence (both red and green), we first performed control experiments to ensure that we could distinguish solved and unsolved colonies. To test fluorescence phenotypes when both split GFP and split RFP are the same cell, we used our positive control constructs ([http://partsregistry.org/Part:BBa_I715045 HPP<sub>0</sub>] and [http://partsregistry.org/Part:BBa_I715042 HPP-A<sub>0</sub>]) with split RFP and split GFP both downstream of the T7 polymerase promoter. In the presence of T7 polymerase, cells with the split RFP/GFP plasmid should demonstrate both green and red fluorescence. <br />
<br />
We first tried cotransforming our HPP<sub>0</sub> construct with a T7 RNA polymerase plasmid construct ([http://partsregistry.org/Part:BBa_I715038 BBa_I715038]). As can be seen in [https://2007.igem.org/Image:SplitRFP-GFP.jpg Figure 3] below, this cotransformation resulted in bright green colonies that demonstrated minimal red fluorescence. When we measured the fluorescence of the cotransformed control, RFP was not present at detectable levels (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
We then tried transforming our HPP-A<sub>0</sub> control plasmid into cells that already contained T7 polymerase in their chromosome (T7 RNAP cells). As can be seen in [https://2007.igem.org/Image:DSC02737.jpeg Figure 4] below, these colonies displayed the yellowish color we expected. Based on our fluorescence data, (shown [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]), we were able to show that GFP and RFP were present at detectable levels in this control. We hypothesize that the difference in the two controls is due to higher concentrations of T7 RNA polymerase in the T7 RNAP cells than in the cotransformants. We therefore used the T7 RNAP cells for future experiments.<br />
<br />
[[Image:SplitRFP-GFP.jpg|thumb|left|300px|'''Figure 3''': Colonies containing both the T7 RNA polymerase plasmid and the HPP<sub>0</sub> control plasmid demonstrated strong green fluorescence, but minimal red fluorescence.]]<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|380px|'''Figure 4''': T7 RNAP colonies that contain the HPP-A<sub>0</sub> control plasmid demonstrated the expected yellow fluorescence.]]<br />
[[Image:Linebreak.png]]<br />
<br />
In addition to the regular split RFP/GFP controls, we also tested 2 "hybrid" constructs to ensure that they would not fluoresce. GFP and RFP have similar structures and functions. Previous studies have shown that it is possible to modify GFP to display a wide range of color phenotypes. We created the following parts: Plac-RBS-RFP1-hixC-GFP2 ([http://partsregistry.org/Part:BBa_I715036 BBa_I715036]) and Plac-RBS-GFP1-hixC-RFP2 ([http://partsregistry.org/Part:BBa_I715035 BBa_I715035]). A plasmid may, at some point during its flipping process, contain such sequences. We tested to make sure that the similarity of these two proteins did not make them compatible enough to fluoresce. It was found that neither of these parts show any fluorescence or color change (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Dectection of Fluoresence by Spectroscopy==<br />
We used a fluorometer to measure the fluorescence of various HPP constructs. First, we had to determine the excitation and emission wavelengths that would allow us to detect the green and red fluorescence of split genes. Scans were conducted in order to find these wavelengths. The graphs below show scans of excitation wavelengths that produce fluorescence at an emission wavelength of 515 nm , the wavelength we found to be best for green, and 608 nm, the best wavelength for red. We used these scans to pick a peak emission wavelength for further testing. <br />
<br />
For these scans we used the following constructs:<br><br />
'''pLac-RBS-GFP1-''hixC''-GFP2''' - This construct fluoresces green on the UV box.<br><br />
'''HPP-A<sub>0</sub>''' - This construct fluoresces yellow on the UV box.<br><br />
'''HPP-A<sub>1</sub>''' - This construct fluoresces red on the UV box.<br><br />
'''HPP-A<sub>2</sub>''' - This construct shows no fluorescence on the UV box.<br><br />
<br><br />
<br />
[[Image:GFP Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 515 nm (green light)]]<br />
<br />
[[Image:Red Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 608 nm (red light)]]<br />
<br />
<br><br />
On the basis of these scans, we used excitation/emission wavelengths of 450 nm / 515 nm for green and 560 nm / 608 nm for red to obtain the results shown below.<br />
<br><br />
<br />
===<center>Normalized* Fluorimetry Results</center>===<br />
{| border="1" cellpadding="5" cellspacing="0" align="center" width="100%"<br />
|-<br />
! style="color: black; background-color: white;" | Construct<br />
! style="color: black; background-color: white;" | Fluorescent Color on UV Box<br />
! style="color: black; background-color: white;" | Green<br />
! style="color: black; background-color: white;" | Red<br />
|-<br />
<br />
|pLac-RBS-<span style="color:green">GFP</span><br />
|None<br />
|9**<br />
|2<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span><br />
|Red<br />
|7<br />
|<span style="color:red">263</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span>-RBS-<span style="color:green">GFP</span><br />
|Red<br />
|<span style="color:green">144</span><br />
|<span style="color:red">370</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|Green<br />
|<span style="color:green">136</span><br />
|0<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|0<br />
|<span style="color:red">147</span><br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|11<br />
|2<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|None<br />
|13<br />
|2<br />
|-<br />
<br />
|HPP<sub>0</sub>***<br />
|Green<br />
|<span style="color:green">72</span><br />
|18<br />
|-<br />
<br />
|HPP-A<sub>0</sub><br />
|Yellow<br />
|<span style="color:green">340</span><br />
|<span style="color:red">255</span><br />
|-<br />
<br />
|HPP-A<sub>1</sub><br />
|Red<br />
|1<br />
|<span style="color:red">143</span><br />
|-<br />
<br />
|HPP-A<sub>2</sub><br />
|None<br />
|11<br />
|3<br />
|-<br />
<br />
<br />
|HPP-B<sub>1</sub>***<br />
|hybrid green<br />
|15<br />
|3<br />
|-<br />
<br />
|}<br />
'* To normalize the data, 57 fluorescence units were subtracted from the Green column and 18 from the Red column. These were the lowest fluorescence values for each wavelength.<br><br />
'** We hypothesize that the scan of this construct was faulty, as the cells fluoresce bright green under the UV lamp.<br><br />
'*** These 2 constructs were cotransformed with the T7 RNA polymerase plasmid (as opposed to being transformed into T7 RNAP cells). This could explain their reduced fluorescence.<br />
<br />
<br><br />
<br />
==Flipping Detection by Phenotype==<br />
<br />
To test for flipping by phenotype, we used our 3 - [[Davidson_Missouri_W/Results#Constructs |Graph A]] constructs from above, HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>. As expected, unflipped HPP-A<sub>0</sub> fluoresced yellow, unflipped HPP-A<sub>1</sub> fluoresced red, and unflipped HPP-A<sub>2</sub> demonstrated no fluorescence when transformed into the T7 RNAP cells (See Figures 5-7 below).<br />
<br />
A fragment containing the Hin expression cassette (pLac-RBS-Hin-LVA, [http://partsregistry.org/Part:BBa_S03536 BBa_S03536]) was ligated in front of each of the 3 HPP-A constructs. These HPP-A + Hin plasmids were then transformed into T7 RNAP cells. A colony from each transformation was picked and grown overnight for plasmid mini-prep. Each of the 3 constructs was restriction digested and the insert sizes were verified to be correct. The 3 constructs were then retransformed into T7 RNAP cells, and the resulting transformation mixture was streaked for colony isolation. '''The HPP-A<sub>0</sub> + Hin plate contained mostly yellow colonies but also red and green (See Figures 8 and 11). The Hin HPP-A<sub>1</sub> + Hin plate contained red, green and yellow colonies (See Figures 9 and 12). The HPP-A<sub>2</sub> + Hin plate contained mostly nonfluorescent colonies with a few green and red colonies (See Figures 10 and 13).'''<br><br />
<br />
<br />
''Click twice on the images below for higher resolution.''<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|270px|'''Figure 5:''' HPP-A<sub>0</sub> - These colonies fluoresced yellow]] [[Image:DSC02738.jpeg|thumb|left|270px|'''Figure 6:''' HPP-A<sub>1</sub> - These colonies fluoresced red]] [[Image:DSC02739.jpeg|thumb|left|270px|'''Figure 7:''' HPP-A<sub>2</sub> - These colonies do not fluoresce]]<br />
<br />
[[Image:DSC02746.jpeg|thumb|left|270px|'''Figure 8:''' HPP-A<sub>0</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02744.jpeg|thumb|left|270px|'''Figure 9:''' HPP-A<sub>1</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02736.jpeg|thumb|left|270px|'''Figure 10:''' HPP-A<sub>2</sub> + Hin - The addition of Hin has produced green and red colonies]]<br />
<br />
[[Image:Hin-ABC-cropped2.JPG|thumb|left|270px|'''Figure 11:''' HPP-A<sub>0</sub> + Hin - closeup of Figure 8]] <br />
[[Image:Hin-ACB-cropped3.JPG|thumb|left|270px|'''Figure 12:''' HPP-A<sub>1</sub> + Hin - closeup of Figure 9]]<br />
[[Image:Hin-BAC-cropped.JPG|thumb|left|270px|'''Figure 13:''' HPP-A<sub>2</sub> + Hin - closeup of Figure 10]]<br />
[[Image:Linebreak.png]]<br />
[[Image:Hin-ABC-10-26.JPG|thumb|left|270px|'''Figure 14:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-10-26.JPG|thumb|left|270px|'''Figure 15:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-10-26.JPG|thumb|left|270px|'''Figure 16:''' HPP-A<sub>2</sub> + Hin]]<br />
<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Flipping Detection by PCR==<br />
<br />
In order to provide physical evidence of Hin-mediated DNA rearrangement of our HPP-A + Hin constructs, we designed PCR primers that would allow us to determine the position of the GFP2 gene half. The experiment below shows the results of PCR using the universal reverse primer [http://partsregistry.org/Part:BBa_G00101 G00101], which binds to the 3' biobrick suffix, and a GFP2-forward primer. <br><br><br />
Here is how the primers bind to the 3 HPP-A constructs:<br><br />
[[Image:HPP-A_PCR_Primers.png|frame|none|400px|'''PCR Primer Binding''': (from top to bottom) HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>]]<br><br />
<br><br />
[[Image:PCR 10-25-07.JPG|thumb|left|270px|Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000 bp), Lanes 2 : HPP-A<sub>0</sub>, Lanes 3 : HPP-A<sub>1</sub>, Lanes 4 : HPP-A<sub>2</sub>, Lanes 5 : Hin+HPP-A<sub>0</sub>, Lanes 6 : Hin+HPP-A<sub>1</sub>, Lanes 7 : Hin+HPP-A<sub>2</sub>]]<br />
<br><br />
[[Image:gels_10_25_07.JPG|thumb|left|270px|'''Left Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000 bp), Lanes 2,3 : ClaI digest of Hin+HPP-A<sub>0</sub>, Lanes 4,5 : ClaI digest of Hin+HPP-A<sub>1</sub>, Lanes 6,7 : ClaI digest of Hin+HPP-A<sub>2</sub><br><br />
'''Right Gel''' - Lane 2 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000 bp), Lane 3 : EcoRI+PstI digest of Hin+HPP-A<sub>0</sub>, Lanes 5 : EcoRI+PstI digest of Hin+HPP-A<sub>1</sub>, Lanes 7 : EcoRI+PstI digest of Hin+HPP-A<sub>2</sub>]]<br />
<br />
<br />
<br><br />
[[Image:Linebreak.png]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Conclusions==<br />
We were able to successfully insert a 13 amino acid ''hixC'' site into Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), maintaining the functionality of the proteins and allowing for flipping of the DNA by Hin Recombinase. When both split RFP and split GFP are present in the same cell, a yellow color can be observed by eye under UV light, and both red and green fluorescence are detected by fluorimetry. We thus attempted to construct a simple bacterial computer capable of solving a 3-node Hamiltonian Path Problem. We planned to select for the solution by picking cells that fluoresced yellow (both red and green).<br />
<br />
To test for flipping of our HPP computer constructs, we attatched a Hin-expression vector to the HPP construct and observed phenotypic differences in colonies. For all of the starting orientations, we observed colonies of varying colors, suggesting that some colonies had flipped away from their starting orientation and into a new orientation that produced a different phenotype. We also tested for flipping by PCR and were able to see multiple bands for all of our flipped constructs. These results strongly suggest that flipping has occured in each of our HPP constructs. <br />
<br />
In the coming days, we plan to retransform our flipped HPP plasmid (without Hin-Recombinase) into T7 RNAP cells in order to obtain clonal colonies. We hope that selecting for a yellow colony and sequencing this plasmid would give us the solution to the Hamiltonian Path Problem that was programmed into the cells. <br />
<br />
<br><br />
<hr><br />
<center><br />
<[[Davidson Missouri W/Gene splitting | Previous Section]] | [[Davidson Missouri W/Traveling Salesperson Problem | Next Section>]]<br />
</center></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_W/ResultsDavidson Missouri W/Results2007-10-26T17:46:12Z<p>Macampbell: /* Flipping Detection by PCR */</p>
<hr />
<div><center>[[Davidson Missouri W| <span style="color:red">Home</span>]] | [[Davidson Missouri W/Background Information| <span style="color:red">Background Information</span>]] | [[Davidson Missouri W/Solving the HPP in vivo| <span style="color:red">Current Project: Solving the Hamiltonian Path Problem ''in vivo''</span>]] | [[Davidson Missouri W/Mathematical Modeling| <span style="color:red">Mathematical Modeling</span>]] | [[Davidson Missouri W/Gene splitting| <span style="color:red">Gene Splitting</span>]] | [[Davidson Missouri W/Results| <span style="color:black">Results</span>]] | [[Davidson Missouri W/Traveling Salesperson Problem| <span style="color:red">Traveling Salesperson Problem</span> ]] | [[Davidson Missouri W/Software|<span style="color:red">Software</span>]] | [[Davidson Missouri W/Resources and Citations|<span style="color:red">Resources and Citations</span>]]</center><br />
<br />
<hr><br />
<br><br />
<br />
==Gene Splitting==<br />
We were able to split two genes: GFP and RFP. Split GFP has strong green fluorescence. Split RFP's red color is much reduced compared to wild-type RFP. It takes overnight incubation at room temperature for the red color to be visible in white light. Both colors are fluorescent under UV light, although the green color predominates. In [https://2007.igem.org/Image:GFPplates.jpg Figure 1] below, a negative-control (on the left) does not fluoresce, but split GFP (on the right) does. [https://2007.igem.org/Image:Split_RFP.png Figure 2] shows a plate of cells containing split RFP.<br />
<br />
[[Image:GFPplates.jpg|thumb|left|500px|'''Figure 1''': The negative control (hixC-GFP2) on the left does not fluoresce, but the experimental split GFP (pLac-RBS-GFP1-hixC-GFP2) on the right does.]]<br />
<br />
[[Image:Split_RFP.png|thumb|left|250px|'''Figure 2''': Cells containing split RFP (pLac-RBS-RFP1-hixC-RFP2) after overnight incubation at room temperature.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Graphs==<br />
<br />
Once we managed to split two genes, we proceeded to implement two different 3-node graphs into plasmids. These graphs were named Graphs A and B and are shown below. For each graph, we wanted to find a Hamiltonian Path from the node represented by RFP (colored in green) to the node represented by the transcriptional terminator (colored in red). The plasmid representation of Graph A is not be capable of flipping into a [https://2007.igem.org/Davidson_Missouri_W/Mathematical_Modeling#True_Positives_and_False_Positives false positive] orientation, while the plasmid representation of Graph B does provide that possibility.<br />
<br />
[[Image:MWSUgraph.png|thumb|200px|left|'''Graph A''': This graph contains 3-nodes and 3-edges. We wanted to find a Hamiltonian path from the node representing RFP (colored in green) to the node representing the transcriptional terminator (colored in red).]] [[Image:DCgraph.png|thumb|200px|none|'''Graph B''': This graph also contains 3-nodes and 3-edges, however it provides the possibility of a false positive solution.]]<br />
[[Image:Linebreak.png]]<br />
<br />
==Starting Orientations==<br />
We constructed the following Hamiltonian Path Problem (HPP) constructs to test our bacterial computer system for Graphs A and B. Constructs that are built to solve [https://2007.igem.org/Image:MWSUgraph.png Graph A] are labeled HPP-A. Constructs that are built to solve [https://2007.igem.org/Image:DCgraph.png Graph B] are labeled HPP-B. A subscript equal to 0 denotes a plasmid in a solved orientation. A subscript that is greater than 0 denotes a plasmid that is in an unordered starting orientation.<br />
<br />
*'''HPP<sub>0</sub> (Positive Control for HPP-A and HPP-B constructs) '''<br>[[Image:Picture_1.png]]<br><br />
*'''HPP-A<sub>0</sub> (Solved Orientation)'''<br>[[Image:Picture_2.png]]<br><br />
*'''HPP-A<sub>1</sub> '''<br>[[Image:Picture_3.png]]<br><br />
*'''HPP-A<sub>2</sub> '''<br>[[Image:Picture_4.png]]<br><br />
*'''HPP-B<sub>1</sub> '''<br>[[Image:Picture_5.png]]<br><br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Controls==<br />
Because are graphs require that solved colonies be selected for by double fluorescence (both red and green), we first performed control experiments to ensure that we could distinguish solved and unsolved colonies. To test fluorescence phenotypes when both split GFP and split RFP are the same cell, we used our positive control constructs ([http://partsregistry.org/Part:BBa_I715045 HPP<sub>0</sub>] and [http://partsregistry.org/Part:BBa_I715042 HPP-A<sub>0</sub>]) with split RFP and split GFP both downstream of the T7 polymerase promoter. In the presence of T7 polymerase, cells with the split RFP/GFP plasmid should demonstrate both green and red fluorescence. <br />
<br />
We first tried cotransforming our HPP<sub>0</sub> construct with a T7 RNA polymerase plasmid construct ([http://partsregistry.org/Part:BBa_I715038 BBa_I715038]). As can be seen in [https://2007.igem.org/Image:SplitRFP-GFP.jpg Figure 3] below, this cotransformation resulted in bright green colonies that demonstrated minimal red fluorescence. When we measured the fluorescence of the cotransformed control, RFP was not present at detectable levels (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
We then tried transforming our HPP-A<sub>0</sub> control plasmid into cells that already contained T7 polymerase in their chromosome (T7 RNAP cells). As can be seen in [https://2007.igem.org/Image:DSC02737.jpeg Figure 4] below, these colonies displayed the yellowish color we expected. Based on our fluorescence data, (shown [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]), we were able to show that GFP and RFP were present at detectable levels in this control. We hypothesize that the difference in the two controls is due to higher concentrations of T7 RNA polymerase in the T7 RNAP cells than in the cotransformants. We therefore used the T7 RNAP cells for future experiments.<br />
<br />
[[Image:SplitRFP-GFP.jpg|thumb|left|300px|'''Figure 3''': Colonies containing both the T7 RNA polymerase plasmid and the HPP<sub>0</sub> control plasmid demonstrated strong green fluorescence, but minimal red fluorescence.]]<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|380px|'''Figure 4''': T7 RNAP colonies that contain the HPP-A<sub>0</sub> control plasmid demonstrated the expected yellow fluorescence.]]<br />
[[Image:Linebreak.png]]<br />
<br />
In addition to the regular split RFP/GFP controls, we also tested 2 "hybrid" constructs to ensure that they would not fluoresce. GFP and RFP have similar structures and functions. Previous studies have shown that it is possible to modify GFP to display a wide range of color phenotypes. We created the following parts: Plac-RBS-RFP1-hixC-GFP2 ([http://partsregistry.org/Part:BBa_I715036 BBa_I715036]) and Plac-RBS-GFP1-hixC-RFP2 ([http://partsregistry.org/Part:BBa_I715035 BBa_I715035]). A plasmid may, at some point during its flipping process, contain such sequences. We tested to make sure that the similarity of these two proteins did not make them compatible enough to fluoresce. It was found that neither of these parts show any fluorescence or color change (See fluorescence data [https://2007.igem.org/Davidson_Missouri_W/Results#Dectection_of_Fluoresence_by_Spectroscopy below]).<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Dectection of Fluoresence by Spectroscopy==<br />
We used a fluorometer to measure the fluorescence of various HPP constructs. First, we had to determine the excitation and emission wavelengths that would allow us to detect the green and red fluorescence of split genes. Scans were conducted in order to find these wavelengths. The graphs below show scans of excitation wavelengths that produce fluorescence at an emission wavelength of 515 nm , the wavelength we found to be best for green, and 608 nm, the best wavelength for red. We used these scans to pick a peak emission wavelength for further testing. <br />
<br />
For these scans we used the following constructs:<br><br />
'''pLac-RBS-GFP1-''hixC''-GFP2''' - This construct fluoresces green on the UV box.<br><br />
'''HPP-A<sub>0</sub>''' - This construct fluoresces yellow on the UV box.<br><br />
'''HPP-A<sub>1</sub>''' - This construct fluoresces red on the UV box.<br><br />
'''HPP-A<sub>2</sub>''' - This construct shows no fluorescence on the UV box.<br><br />
<br><br />
<br />
[[Image:GFP Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 515 nm (green light)]]<br />
<br />
[[Image:Red Fluorescence.png|frame|none|Excitation Spectra at an Emission Wavelength of 608 nm (red light)]]<br />
<br />
<br><br />
On the basis of these scans, we used excitation/emission wavelengths of 450 nm / 515 nm for green and 560 nm / 608 nm for red to obtain the results shown below.<br />
<br><br />
<br />
===<center>Normalized* Fluorimetry Results</center>===<br />
{| border="1" cellpadding="5" cellspacing="0" align="center" width="100%"<br />
|-<br />
! style="color: black; background-color: white;" | Construct<br />
! style="color: black; background-color: white;" | Fluorescent Color on UV Box<br />
! style="color: black; background-color: white;" | Green<br />
! style="color: black; background-color: white;" | Red<br />
|-<br />
<br />
|pLac-RBS-<span style="color:green">GFP</span><br />
|None<br />
|9**<br />
|2<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span><br />
|Red<br />
|7<br />
|<span style="color:red">263</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP</span>-RBS-<span style="color:green">GFP</span><br />
|Red<br />
|<span style="color:green">144</span><br />
|<span style="color:red">370</span><br />
|-<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|Green<br />
|<span style="color:green">136</span><br />
|0<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|0<br />
|<span style="color:red">147</span><br />
|-<br />
<br />
<br />
|pLac-RBS-<span style="color:green">GFP1</span>-''hixC''-<span style="color:red">RFP2</span><br />
|None<br />
|11<br />
|2<br />
|-<br />
<br />
|pLac-RBS-<span style="color:red">RFP1</span>-''hixC''-<span style="color:green">GFP2</span><br />
|None<br />
|13<br />
|2<br />
|-<br />
<br />
|HPP<sub>0</sub>***<br />
|Green<br />
|<span style="color:green">72</span><br />
|18<br />
|-<br />
<br />
|HPP-A<sub>0</sub><br />
|Yellow<br />
|<span style="color:green">340</span><br />
|<span style="color:red">255</span><br />
|-<br />
<br />
|HPP-A<sub>1</sub><br />
|Red<br />
|1<br />
|<span style="color:red">143</span><br />
|-<br />
<br />
|HPP-A<sub>2</sub><br />
|None<br />
|11<br />
|3<br />
|-<br />
<br />
<br />
|HPP-B<sub>1</sub>***<br />
|Darker green<br />
|15<br />
|3<br />
|-<br />
<br />
|}<br />
'* To normalize the data, 57 fluorescence units were subtracted from the Green column and 18 from the Red column. These were the lowest fluorescence values for each wavelength.<br><br />
'** We hypothesize that the scan of this construct was faulty, as the cells fluoresce bright green under the UV lamp.<br><br />
'*** These 2 constructs were cotransformed with the T7 RNA polymerase plasmid (as opposed to being transformed into T7 RNAP cells). This could explain their reduced fluorescence.<br />
<br />
<br><br />
<br />
==Flipping Detection by Phenotype==<br />
<br />
To test for flipping by phenotype, we used our 3 - [[Davidson_Missouri_W/Results#Constructs |Graph A]] constructs from above, HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>. As expected, unflipped HPP-A<sub>0</sub> fluoresced yellow, unflipped HPP-A<sub>1</sub> fluoresced red, and unflipped HPP-A<sub>2</sub> demonstrated no fluorescence when transformed into the T7 RNAP cells (See Figures 5-7 below).<br />
<br />
A fragment containing the Hin expression cassette (pLac-RBS-Hin-LVA, [http://partsregistry.org/Part:BBa_S03536 BBa_S03536]) was ligated in front of each of the 3 HPP-A constructs. These HPP-A + Hin plasmids were then transformed into T7 RNAP cells. A colony from each transformation was picked and grown overnight for plasmid mini-prep. Each of the 3 constructs was restriction digested and the insert sizes were verified to be correct. The 3 constructs were then retransformed into T7 RNAP cells, and the resulting transformation mixture was streaked for colony isolation. '''The HPP-A<sub>0</sub> + Hin plate contained mostly yellow colonies but also red and green (See Figures 8 and 11). The Hin HPP-A<sub>1</sub> + Hin plate contained red, green and yellow colonies (See Figures 9 and 12). The HPP-A<sub>2</sub> + Hin plate contained mostly nonfluorescent colonies with a few green and red colonies (See Figures 10 and 13).'''<br><br />
<br />
<br />
''Click twice on the images below for higher resolution.''<br />
<br />
[[Image:DSC02737.jpeg|thumb|left|270px|'''Figure 5:''' HPP-A<sub>0</sub> - These colonies fluoresced yellow]] [[Image:DSC02738.jpeg|thumb|left|270px|'''Figure 6:''' HPP-A<sub>1</sub> - These colonies fluoresced red]] [[Image:DSC02739.jpeg|thumb|left|270px|'''Figure 7:''' HPP-A<sub>2</sub> - These colonies do not fluoresce]]<br />
<br />
[[Image:DSC02746.jpeg|thumb|left|270px|'''Figure 8:''' HPP-A<sub>0</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02744.jpeg|thumb|left|270px|'''Figure 9:''' HPP-A<sub>1</sub> + Hin - The addition of Hin has produced green, red, and yellow colonies]] [[Image:DSC02736.jpeg|thumb|left|270px|'''Figure 10:''' HPP-A<sub>2</sub> + Hin - The addition of Hin has produced green and red colonies]]<br />
<br />
[[Image:Hin-ABC-cropped2.JPG|thumb|left|270px|'''Figure 11:''' HPP-A<sub>0</sub> + Hin - closeup of Figure 8]] <br />
[[Image:Hin-ACB-cropped3.JPG|thumb|left|270px|'''Figure 12:''' HPP-A<sub>1</sub> + Hin - closeup of Figure 9]]<br />
[[Image:Hin-BAC-cropped.JPG|thumb|left|270px|'''Figure 13:''' HPP-A<sub>2</sub> + Hin - closeup of Figure 10]]<br />
[[Image:Linebreak.png]]<br />
[[Image:Hin-ABC-10-26.JPG|thumb|left|270px|'''Figure 14:''' HPP-A<sub>0</sub> + Hin]]<br />
[[Image:Hin-ACB-10-26.JPG|thumb|left|270px|'''Figure 15:''' HPP-A<sub>1</sub> + Hin]]<br />
[[Image:Hin-BAC-10-26.JPG|thumb|left|270px|'''Figure 16:''' HPP-A<sub>2</sub> + Hin]]<br />
<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Flipping Detection by PCR==<br />
<br />
In order to provide physical evidence of Hin-mediated DNA rearrangement of our HPP-A + Hin constructs, we designed PCR primers that would allow us to determine the position of the GFP2 gene half. The experiment below shows the results of PCR using the universal reverse primer [http://partsregistry.org/Part:BBa_G00101 G00101], which binds to the 3' biobrick suffix, and a GFP2-forward primer. <br><br><br />
Here is how the primers bind to the 3 HPP-A constructs:<br><br />
[[Image:HPP-A_PCR_Primers.png|frame|none|400px|'''PCR Primer Binding''': (from top to bottom) HPP-A<sub>0</sub>, HPP-A<sub>1</sub>, HPP-A<sub>2</sub>]]<br><br />
<br><br />
[[Image:PCR 10-25-07.JPG|thumb|left|270px|Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000 bp), Lanes 2 : HPP-A<sub>0</sub>, Lanes 3 : HPP-A<sub>1</sub>, Lanes 4 : HPP-A<sub>2</sub>, Lanes 5 : Hin+HPP-A<sub>0</sub>, Lanes 6 : Hin+HPP-A<sub>1</sub>, Lanes 7 : Hin+HPP-A<sub>2</sub>]]<br />
<br><br />
[[Image:gels_10_25_07.JPG|thumb|left|270px|'''Left Gel''' - Lane 1 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000 bp), Lanes 2,3 : ClaI digest of Hin+HPP-A<sub>0</sub>, Lanes 4,5 : ClaI digest of Hin+HPP-A<sub>1</sub>, Lanes 6,7 : ClaI digest of Hin+HPP-A<sub>2</sub><br><br />
'''Right Gel''' - Lane 2 : MW marker (50, 100, 200, 300, 400, 500, 750, 1000, 1400/1550, 2000, 3000, 4000 bp), Lane 3 : EcoRI+PstI digest of Hin+HPP-A<sub>0</sub>, Lanes 5 : EcoRI+PstI digest of Hin+HPP-A<sub>1</sub>, Lanes 7 : EcoRI+PstI digest of Hin+HPP-A<sub>2</sub>]]<br />
<br />
<br />
<br><br />
[[Image:Linebreak.png]]<br />
<br />
[[Image:Linebreak.png]]<br />
<br />
==Conclusions==<br />
We were able to successfully insert a 13 amino acid ''hixC'' site into Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), maintaining the functionality of the proteins and allowing for flipping of the DNA by Hin Recombinase. When both split RFP and split GFP are present in the same cell, a yellow color can be observed by eye under UV light, and both red and green fluorescence are detected by fluorimetry. We thus attempted to construct a simple bacterial computer capable of solving a 3-node Hamiltonian Path Problem. We planned to select for the solution by picking cells that fluoresced yellow (both red and green).<br />
<br />
To test for flipping of our HPP computer constructs, we attatched a Hin-expression vector to the HPP construct and observed phenotypic differences in colonies. For all of the starting orientations, we observed colonies of varying colors, suggesting that some colonies had flipped away from their starting orientation and into a new orientation that produced a different phenotype. We also tested for flipping by PCR and were able to see multiple bands for all of our flipped constructs. These results strongly suggest that flipping has occured in each of our HPP constructs. <br />
<br />
In the coming days, we plan to retransform our flipped HPP plasmid (without Hin-Recombinase) into T7 RNAP cells in order to obtain clonal colonies. We hope that selecting for a yellow colony and sequencing this plasmid would give us the solution to the Hamiltonian Path Problem that was programmed into the cells. <br />
<br />
<br><br />
<hr><br />
<center><br />
<[[Davidson Missouri W/Gene splitting | Previous Section]] | [[Davidson Missouri W/Traveling Salesperson Problem | Next Section>]]<br />
</center></div>Macampbellhttp://2007.igem.org/wiki/index.php/Jamboree/Jamboree_handbookJamboree/Jamboree handbook2007-10-26T15:53:02Z<p>Macampbell: /* Team Boxes */</p>
<hr />
<div>{{TOCright}}<br />
<br />
===iGEM 2007 Jamboree Handbook===<br />
<br />
Welcome to the iGEM 2007 Jamboree! The next few days will be full of exciting presentations, stimulating conversations, well-deserved awards, and most of all, a lot of fun. The following information will guide you through the whole Jamboree event from what to expect at Friday night set-up all the way to where you can pick up your lunch on Sunday afternoon after the awards ceremony. Please read through this whole guide! It contains a lot of useful instructions that will make your Jamboree experience go as smoothly as possible. And as a special note to team leaders, you will have some extra responsibilities so make sure you are aware of what you are expected to do.<br />
<br />
If you have a question at any point during the Jamboree, just look for one of the iGEM staff members in the bright yellow polo shirts [IMAGE]<br />
<br />
<br />
Note: For all locations on the Stata Center - Student Street, consult the map in your folder or [https://2007.igem.org/Image:Stata_street_map.jpg '''see the map here'''].<br />
<br />
You can see a photo of the Stata Center [http://whereis.mit.edu/map-jpg?selection=32&Buildings=go '''here''']<br />
<br />
<br />
<br />
====Registration/Check-In====<br />
<br />
Teams can check-in Friday night (November 2) at Jamboree pre-registration beginning at 6pm. It will take place at the MIT Stata Center. The street address is 32 Vassar Street, Cambridge, MA 02139. <br />
<br />
Regular registration begins Saturday (November 3) at 8:00am, also in the Stata Center. If you did not register online, it will be possible to register on-site on Saturday morning.<br />
<br />
At registration you will pick up your team box containing badges, team member registration folders, team member participation certificates, and team seals of achievement. Each team leader (or a designated representative) will be responsible for picking up the team box. This means that each member of the team DOES NOT have to stand in line at Registration. Important: in order to pick up your team box, you will be required to turn in all [[Jamboree/Release_Form|'''photo release forms''']] for each person that is on your team roster. The release forms are absolutely necessary (see section on release forms below). Registration personnel will check off each release form for each team member. Badge(s) pertaining to each person missing a release form will be held at Registration until the form has been turned in. Badges will be necessary for entrance into presentation rooms and for access to food. <br />
<br />
<br />
====Badges====<br />
<br />
You will receive your name badge as part of your team box, as long as you have submitted your photo release form. Please wear your badge at all times during the Jamboree and make sure it is clearly visible. Badges will be necessary for entrance into presentation rooms and for access to food. If you do not have a badge, you must register in order to obtain one. <br />
<br />
<br />
====Friday Night Setup====<br />
<br />
Teams will be allowed to set-up on the Friday night (November 2) prior to the Jamboree beginning at 6:00pm. You can put up your poster, see the layout of your presentation room, practice your presentation, and get to know fellow iGEM members. Note that there will not be technical staff on hand to help with audio/visual equipment. Please leave all presentation rooms in the condition that you found them. There will also be some food provided on a first come, first serve basis. You can find a [[Jamboree/Schedule/Friday/Practice talks|'''sign up sheet here''']].<br />
<br />
Please keep in mind that Friday night setup will only run from 6:00pm to 10:00pm. See the Security section below for detailed security information. <br />
<br />
<br />
====Photo Release====<br />
<br />
One of the products of the iGEM 2006 Jamboree was the [https://static.igem.org/mediawiki/2006/9/9f/IGEM_From_Above-community2.png '''iGEM from Above'''] photograph. The use of this photograph, and all others from the 2006 Jamboree, was made possible due to the existence of a filled-out photo release form from every person attending the Jamboree. We must do the same for the 2007 Jamboree. In order to comply with the law, every person attending the Jamboree must fill out a photo release form. There is a [[Jamboree/Release_Form|'''copy of the release form''']] on the iGEM 07 wiki that you should print, fill out, and bring with you to the Jamboree. Note: Team leaders, you must have a filled-out photo release form for each member of your team that is on the official roster before you can pick up your team member badges and team box. There will also be blank copies available at registration for you to fill out if you need another copy. If you have any questions or need further clarification, feel free to ask an iGEM staff member (in the yellow iGEM polo shirts). <br />
<br />
<br />
====Team Leaders====<br />
<br />
As mentioned above, team leaders have a particular responsibility. Each team leader (or a designated representative) will be responsible for gathering all team member photo release forms and handing them in at registration in order to obtain your team box. <br />
<br />
<br />
====Team Boxes====<br />
<br />
Your team box will contain the following:<br />
<br />
* Team member badges<br />
* Team member certificates<br />
* Team member folders (with Jamboree material)<br />
* Team seals of achievement<br />
<br />
Notes: Team leaders are responsible for picking up team boxes. Badges are to be worn at all times.<br />
<br />
====Seals of Achievement====<br />
<br />
This year we are also designating achievements for documentation of your project and contributions to the synthetic biology community. These achievements are signified by either a bronze, silver, or gold seal, which each team member can adhere to their participatory certificate. These designations will be based on their performance in the [[Jamboree/Compete#Online_Judging_Process|'''online judging''']] round (online judging occurs in the week directly before the Jamboree).<br />
<br />
Each task below gives you one (1) point:<br />
* Documentation of project on the iGEM 07 wiki<br />
* Submission of physical DNA for parts<br />
* Documentation of parts in the Registry.<br />
<br />
<br />
To win an award you need to have the following point counts:<br />
* Bronze award: One (1) point total<br />
* Silver award: Two (2) points total<br />
* Gold award: Three (3) points total<br />
<br />
<br />
You will be handed your team seals of achievement (one for each member on the official roster) when you pick up your team box at Registration. It is intended that your seal be placed on your participation certificate (for each team member) as well as one on your team poster.<br />
<br />
====Posters====<br />
<br />
Each team is required to [[Jamboree/Compete#Project_Poster|'''present a poster''']] at the Jamboree. The poster must be 4 ft (48 in, 121.92 cm) by 4 ft (48 in, 121.92cm). Each poster should be hung up on one of the poster stands that will be set up on the Stata Center - Student Street. Supplies for putting up your posters will be provided. Poster locations are not assigned for each team. Teams may choose where to put their poster in the locations that are available at that time. <br />
<br />
<br />
====Presentation Room Overflow Areas====<br />
<br />
Overflow areas for each of the 4 smaller presentation rooms (124, 141, 144, 155) have been set up to accommodate participants who wish to watch presentations from outside of the presentation rooms. There will be plasma screens set up with audio speakers. Each overflow area will have adequate seating and electrical outlets. Check the map for detailed locations for each overflow area. <br />
<br />
<br />
====Awards/Frames====<br />
<br />
Awards will be presented at the awards ceremony on Sunday (November 4). Each team that wins an award will get one trophy for the team as well as award certificates for each team member. These certificates are separate from the participation certificates that every team member from every team gets and that can be found in the team box. Both the award certificates and the participation certificates look great when framed. Certificate frames will be provided by iGEM HQ and will be available in the Stata Center from the time the awards ceremony finishes until 2pm.<br />
<br />
<br />
====IGEM Store====<br />
<br />
Come buy some iGEM products at the iGEM store! This year we will be selling iGEM hoodies (sweatshirts) for US$40 and iGEM iron-on patches for US$5. Wear your hoodies on those chilly days to keep you warm and stick your patch on your backpack, lab coat, jacket, or any other place you want to express your love for iGEM! And don't forget to buy some for your family and friends. The iGEM store will accept US dollars and all major credit cards. <br />
<br />
<br />
====Shout About iGEM Kiosk====<br />
<br />
The Jamboree is a great opportunity to share all of the hard work that you have put into your project, not only with the rest of the iGEM community, but with your family and friends as well. We would like to give you the opportunity to contact your friends and family right from the Jamboree and with an official iGEM Jamboree message. There will be a kiosk (with table and chairs) set up on the State Center - Student Street where you will be able to quickly fill out a few fields and send a pre-generated digital postcard to anyone you wish. We will also have a photographer that can take your picture and upload it to the postcard. This way you can send a live message to all of your family and friends. Don't forget to wear your team t-shirt or iGEM hoodie to show off in the photo. To find out more information, visit the ''Shout about iGEM'' kiosk on the student street.<br />
<br />
<br />
====Family and Friends====<br />
<br />
Family and friends are welcome for all iGEM Jamboree events. If non-iGEM team members (e.g. family and friends) want to attend the presentation sessions, poster sessions, and reception/dinner on Saturday, they have to officially register and get a badge (on-site registration is available and can be paid for with all major credit cards, US$275). If family and friends want to attend the awards ceremony (Sunday, November 4), they may do so without registering or obtaining badges. Admission to Kresge Auditorium for the awards ceremony will be free. <br />
<br />
<br />
====Food====<br />
<br />
Food will be provided throughout the Jamboree. Badges must be (visibly) worn in order to have access to all food. <br />
<br />
On Friday there will be pizza and refreshments on a first-come first-serve basis. (Note: badges do not have to be worn on Friday night).<br />
<br />
On Saturday 30 minute coffee breaks occur every 90 minutes, and after the first six presentations and the group photo there will be lunch. Note that there will be three lunch locations on the Stata Center Student Street. Saturday evening after the presentations will be the start of the poster session. There will be a reception with some light food beginning at 5pm '''as well as''' dinner beginning at about 6pm (so save your appetite!). <br />
<br />
On Sunday bagged lunches will be available to eat at Stata after the awards ceremony or take with you if you or your team must leave. Dinner on Sunday is not provided unless you have registered for the Registry Workshop.<br />
<br />
<br />
====Luggage====<br />
<br />
If you need to check out of your hotel on Sunday morning and need to stow your luggage somewhere, you will be able to keep it in Room 124 in the Stata Center. It will be locked and/or supervised during the awards ceremony in Kresge Auditorium. You must pick up your luggage by 2pm. This is absolutely critical. If you do not remove your luggage from Room 124 in the Stata Center by 2pm on Sunday afternoon, it will be moved to another location and you will have to make special arrangements to pick it up. <br />
<br />
<br />
====Transportation====<br />
<br />
The cities of Boston and Cambridge have a public transportation system that is comprised of buses and subways. You can find the website for the [http://www.mbta.com/ '''MBTA here''']. The MIT Stata Center is located on the MBTA Red Line at the Kendall/MIT subway stop. There are also several bus stops in the area which you can see [http://www.mbta.com/rider_tools/servicenearby/?saServiceNearBy=32+Vassar+St.+Cambridge%2C+MA&sLocationServiceNearBy=&selectedPoint=&Hour=2&Minute=&AMPM=PM&sDate=10%2F24%2F2007 '''here''']. <br />
<br />
If you are driving to the Jamboree, you can <br />
[http://whereis.mit.edu/map-jpg?selection=P9&Parking=go '''park in this parking lot''']. Parking is free after 3:00pm on weekdays and all day on weekends.<br />
<br />
<br />
====Security====<br />
<br />
Overnight security will be provided in the Stata Center Friday night. Posters put up on Friday night will be protected by an MIT security officer patrolling the Student Street overnight. Security will not be provided Saturday night.<br />
<br />
<br />
====Contact Information====<br />
<br />
If you need to get in touch with anyone at iGEM headquarters (HQ) for an urgent matter, you may contact the following people:<br />
<br />
* Randy Rettberg +1-978-325-1269<br />
* Meagan Lizarazo +1-917-453-1008<br />
* Mac Cowell +1-231-313-9062<br />
* Isadora Deese +1-617-510-3833<br />
<br />
<br />
====Emergency Information====<br />
<br />
If there is an emergency (medical emergency, fire, police, etc.) please contact MIT campus emergencies numbers:<br />
<br />
* From a campus phone: 100<br />
* From a cell phone, pay phone, or off-campus: +1-617-253-1212</div>Macampbellhttp://2007.igem.org/wiki/index.php/Jamboree/DNA_SubmissionJamboree/DNA Submission2007-09-26T18:16:10Z<p>Macampbell: /* iGEM 2007 Part Submission */</p>
<hr />
<div>{{TOCright}}<br />
<font color=red><small>'''We are still adding information to this page. Feel free to read through it but keep in mind that you should check back often for more updated information!'''</small></font><br />
<br />
==iGEM 2007 Part Submission==<br />
<br />
In order to qualify for winning any part-related awards at the 2007 iGEM Jamboree, the teams must document their parts according to the [https://2007.igem.org/Jamboree/Compete#Part_Documentation_on_the_Registry Part Documentation Guidelines] as well as submit the physical DNA to the Registry. Following these guidelines will begin the part promotion process to get your part elevated to the '''Accepted''' rating level. Only parts that are at the Accepted rating level will be considered for awards. <br />
<br />
For iGEM 2007 we are accepting part submissions in the form of miniprepped DNA. There is an online submission form that must be completed in order to start the DNA submission process (see more details below) [PROVIDE LINK TO FORM]. Each part will go through a physical DNA screening/quality control process, in addition to the part documentation review process, before it is accepted into the Registry. We will also provide online status updates for each part during the submission process so that the part creator can find out at what stage of the screening process their part has gone through.<br />
<br />
<br />
===Part Documentation Guidelines===<br />
All the documentation for your parts is expected to follow the guidelines set by the Judging Committee. The Registry will also provide more elaborate part documentation guidelines that you may want to keep in mind when documenting your parts. Following these expanded guidelines will be necessary in order for parts to start the Part Promotion process and be promoted to Accepted parts. For more information on the Registry Part Promotion process see [http://partsregistry.org/Part_Promotion_Process this Registry page] for a brief explanation. A more detailed explanation of the Part Promotion process will be provided soon. <br />
<br />
<br />
<br />
===Quality Control Process===<br />
In order to assess the quality of the parts accepted into the Registry, we will perform a standard set of experiments with a focus on verifying the length of the insert. In addition we will aim to validate the declared antibiotic resistance of each part.<br />
<br />
The quality control process is comprised of the following tasks:<br />
*'''Transform''': we will transform each sample of DNA into our standard cell strain, Top10<br />
*'''Pick/Inoculate single colony''': after transforming the DNA into Top10 cells, we will pick a single colony and inoculate into the antibiotic specified by the creator. We will also inoculate that colony into the rest of our standard antibiotic set (Ampicillin, Chloramphenicol, Tetracycline, Kanamycin) and watch for growth in the correct antibiotic<br />
*'''Miniprep ''': the bacterial culture grown in the specified antibiotic will be miniprepped to isolate plasmid DNA<br />
*'''Digest ''': the miniprepped plasmid DNA will be cut with EcoRI and PstI to cut out the insert from the plasmid<br />
*'''Gel''': the digested plasmid DNA will be run on an Invitrogen E-gel to visualize the insert and the plasmid. From this gel we can tell whether the part length corresponds to the length designated by the designer. <br />
<br />
If the part length as detected on the E-gel corroborates what the designer has specified in the part documentation AND if the antibiotic testing is consistent with the expected antibiotic growth pattern, the physical DNA for the submitted part will be deemed '''Accepted.'''<br />
<br />
<br />
<br />
===Online Submission Form===<br />
In order to accept physical DNA submission from teams in an organized fashion, we will be requiring teams to complete an online DNA submission form. The form is comprised of four parts:<br />
<br />
====Part Specification page====<br />
<br />
This page is the first page you will come to when you access the online submission form. You will need to specify which one of the four formats you are sending your parts in to the Registry: <br />
[[Image:Minicentrifugetube.jpg|thumb|left|Single PCR tube]]<br />
'''Single PCR tube''' - If you are sending less than 4 samples, you may use the single PCR tube format to send in the physical DNA for your parts. The tube(s) must be wrapped with lab tape with the sequential number of the part written in permanent marker on tab of tape (this number is NOT the part number - it is the number of the sample i.e. #1, #2, #3, #4). When submitting 8-tube strips you must ship them in 50ml Falcon tubes. This format requires a total of 20ng of miniprepped DNA in a final volume of 10ul per sample.<br />
<br><br />
<br><br />
<br><br />
[[Image:Pcrtubes.jpg|thumb|right|8-tube strip]] '''8-tube strip''' - To send more than 4 parts at a time, use the 8-tube strip format. Wrap lab tape around the first tube in the sequence then write the part sequential number (see above) on the lab tape tab. When submitting 8-tube strips you must ship them in 50ml Falcon tubes. Each Falcon tube can hold 2 8-tube strips. ''Using the 8-tube strip format requires a total of 20ng of miniprepped DNA in a final volume of 10ul per sample.'' <br />
<br style="clear:both;"/><br />
'''96-well PCR plate''' - [[Image:96_well_plate.jpg|thumb|right|96-well plate with adhesive foil lid]]If you are submitting a large number of samples you may wish to submit the DNA in 96-well PCR plate formate. You need to add the samples in the correct order. Start with well 1A and work your way down the first column. Then continue on to well 2A and down that column. Proceed in this fashion until you have added all of the samples that you are submitting. Wrap the well 1A with lab tape to provide yourself as well as the folks at the Registry with a visible marker as to where to start processing samples. ''Using this format requires that you submit 20ng of miniprepped DNA in a final volume of 10ul per sample.'' <br />
<br><br />
<br><br />
'''Filter paper grid''' - [[Image:Filter_grid.jpg|thumb|left|Filter paper grid with punching tool]]We have put a lot of effort into testing the use of high quality paper as a method by which to send DNA. The Registry will be sending each team several paper grids onto which they can spot their DNA. You must combine 1.6ul of DNA at 100ng/ul with 0.4ul of 1% Cresol Red dye and spot the resulting 2ul in the middle of the box into which you are spotting your DNA. Begin with box 1A, continue down column 1, continue on to box 2A and down that column. Proceed in this order until you have finished spotting all of the samples that you are submittin. ''You will end up submitting at least 160ng total for each sample.''<br />
<br style="clear:both;"/><br />
<br />
After choosing which method for DNA submission you will use, you will need to enter the Registry part numbers for each sample. Enter the part numbers in the order in which you will be sending the DNA. By default this page will only 8 text fields into which you can input your part numbers. If you are submitting more than 8 parts, click on the appropriate button to '''Add more parts.'''<br />
<br />
Once you have finished entering the part numbers for all of the parts that you are submitting, click on the '''Proceed to fill in part details''' button. This will take you to the next portion of the online submission process.<br />
<br />
====Part Detail page====<br />
<br />
The part detail page is where you will do just that - add all of the details for each of the parts that you will be submitting! There are 5 criteria that we require for each part being submitted. <br />
<br />
*'''Part number''' - As you have already entered the part number on the preliminary part specification page, this field will be automatically filled in for you. Do double-check, however, to make sure that the part numbers that have been filled in correspond to the part numbers for which you are entering the details. <br />
<br />
*'''Plasmid''' - You will be able to choose the plasmid that your part is in from a pull down menu that lists all of the plasmids documented on the Registry. IMPORTANT: If for some reason the plasmid that your part is in does not appear on the pull down menu you must first enter the plasmid into the Registry. Instructions on how to add a plasmid to the Registry can be found HERE. <br />
<br />
*'''Total ng of DNA provided''' - Please provide the total amount of DNA (in ng) that you are submitting to the Registry. This DNA must be miniprepped and we require at least 20ng of DNA in a final volume of 10ul (concentration of 2ng/ul). For filter paper grids we require at least 0.16ug of DNA.<br />
<br />
*'''DNA format''' - While we encourage you to submit DNA in liquid form, you may provide dry DNA if you wish. Please indicate in this field which format you are sending the DNA in. <br />
<br />
*'''Sequenced''' - Please tell us whether you have sequenced this DNA or not. Even you if you haven't sequenced the DNA that you are submitting, the sequence MUST be in the part documentation. See THIS PAGE for the part documentation rules. <br />
<br />
The part detail page is also where you will provide the tracking number for the shipment of parts that you are sending the Registry. This tracking number is mandatory. It allows the ability to see whether the parts that you sent are stuck in customs, therefore delaying your part submission. Please provide the tracking number as well as the carrier that you are using, e.g. DHL 012345678.<br />
<br />
====Part Shipment Review page====<br />
After entering all of your part details you are given the opportunity to review all of the information that you are about to submit to the Registry. Take a look at all of the part details and make sure they are correct. At this point you can either click the Edit button to go back to edit any of the details or press the submit button to finalize your DNA submission. <br />
<br />
====Part Shipment Completion page====<br />
Once you click on the Submit Part Shipment button to finalize your submission you will come to a page that shows that you have successfully submitted your part shipment. You will also see a review of your part details that you entered on the part detail page. At this point you may want to print this page to have a record of the parts that you have submitted. Keep track of your part shipment number and check back to see what stage your submission is in (see below for details). <br />
<br />
<br />
<br />
<br />
===Physical DNA Submission: Submission status===<br />
We want you to be fully aware of the status of your part while it is going through the Registry QC process. For this reason we are creating the ability for you to check back and see what stage your is at. You will be able to see:<br />
<br />
* when we have received the DNA<br />
* when the part has begun the QC process<br />
* when the part has successfully completed the QC process<br />
* if the part did not complete the QC process, you will see when we are waiting for you to send us another sample of the part<br />
<br />
<br />
<br />
===Part Shipping Materials: ordering information===<br />
Single PCR tubes: '''Axygen''' [http://www.axygen.com/jsp/coreIdDetail.jsp?coreId=MCT060 MCT-060-C-S]<br><br />
8-tube strips: '''Axygen''' [http://www.axygen.com/jsp/coreIdDetail.jsp?coreId=PCR0208 PCR-0208-C] or '''BioRad''' [http://www.bio-rad.com/B2B/BioRad/product/br_category.jsp?BV_SessionID=@@@@1422642991.1189446586@@@@&BV_EngineID=ccceaddlmhegdgkcfngcfkmdhkkdflm.0&categoryPath=%2fCatalogs%2fLife+Science+Research%2fAmplification+%7c+PCR%2fPCR+Plastic+Consumables%2fThin-Wall+PCR+Tubes+and+Caps%2fTube+and+Cap+Strips%2fLow-Profile+0.2+ml+Tube+Strips%2f&divName=Corporate&loggedIn=false&lang=English&country=HQ&catLevel=7&catOID=-33540&isPA=false&serviceLevel=Lit+Request&searchStr=TLS+0801&cateName=Ordering+Information TLS 0801]<br><br />
96-well plates: '''VWR''' [https://obi2.vwrsp.com/catalog/product/index.cgi?catalog_number=82006-650&inE=1&highlight=82006-650&from_search=1 82006-650]<br><br />
Filter paper grid: provided by the Registry</div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_W/Project_DescriptionDavidson Missouri W/Project Description2007-08-29T21:12:08Z<p>Macampbell: </p>
<hr />
<div>The 2007 Davidson/Missouri Western (DMW) iGEM team is building an ''E. coli'' computer capable of solving the Hamiltonian Path Problem. ''E. coli'' provide the massive parallel processing power required to solve this computationally intense problem from Graph Theory. We use a Hin/hixC DNA recombination mechanism to randomly generate possible paths through the graph. We then use gene expression and fragment length to screen for a Hamiltonian Path. <br />
<br />
To construct our computer, we have inserted hixC sites into reporter genes in a location that destroys the gene’s function when the split segments are not united but allows for normal gene expression when the two segments are contiguous. So far we have successfully split GFP and RFP in this manner. Our method demands that the Hin/hixC system be able to rearrange multiple DNA elements into every possible ordering. Therefore, we have tested and confirmed that Hin/hixC can rearrange 2 DNA elements into all 8 signed permutations. Our math modeling shows that it will be feasible to detect true Hamiltonian Paths even in complex graphs. We are close to testing our first construct on a simple 3-node graph.</div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-08-29T21:11:30Z<p>Macampbell: /* Project Description */</p>
<hr />
<div>===Davidson & Missouri Western Team Logo, iGEM2007=== <br><br />
<br />
<br />
[[Image:DMW.jpg|thumb|300px|center| ]]<br />
<br><br />
<br />
==[[Davidson Missouri W/Project Description|Project Description]]==<br />
The 2007 Davidson/Missouri Western (DMW) iGEM team is building an ''E. coli'' computer capable of solving the Hamiltonian Path Problem. ''E. coli'' provide the massive parallel processing power required to solve this computationally intense problem from Graph Theory. We use a Hin/hixC DNA recombination mechanism to randomly generate possible paths through the graph. We then use gene expression and fragment length to screen for a Hamiltonian Path. <br />
<br />
To construct our computer, we have inserted hixC sites into reporter genes in a location that destroys the gene’s function when the split segments are not united but allows for normal gene expression when the two segments are contiguous. So far we have successfully split GFP and RFP in this manner. Our method demands that the Hin/hixC system be able to rearrange multiple DNA elements into every possible ordering. Therefore, we have tested and confirmed that Hin/hixC can rearrange 2 DNA elements into all 8 signed permutations. Our math modeling shows that it will be feasible to detect true Hamiltonian Paths even in complex graphs.<br />
<br />
== '''Team Meeting Notes''' ==<br />
<br />
Western Meeting Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/WesternMeetingNotes]<br />
<br />
== '''Math Related Notes''' ==<br />
<br />
Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/MathMeetingNotes]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/FalsePositiveProgram False Positives]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/ProjectsCompleted Projects Completed]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/Graphs Graphs]<br />
<br />
== '''Students''' ==<br />
<br />
[[Image:Logo.gif]]<br />
<br />
• Will DeLoache, Junior Biology Major, [mailto:wideloache@davidson.edu]<br />
<br />
• Oyinade Adefuye, Senior Biology Major, [mailto:oyinadeadefuye@yahoo.com]<br />
<br />
• Jim Dickson, Junior Math and Economics Major, [mailto:jidickson@davidson.edu]<br />
<br />
• Amber Shoecraft, Math Major, [mailto:amshoecraft@davidson.edu]<br />
<br />
• Andrew Martens, Senior Biology Major, [mailto:anmartens@davidson.edu]<br />
<br />
• Michael Waters, Sophomore Biology Major, [mailto:miwaters@davidson.edu]<br />
<br><br />
[[Image:Team1.jpg|200px|]] [[Image:Team_graph.jpg|200px|]]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
<br />
• Jordan Baumgardner, Junior Biology, Biochemistry/Molecular Biology Major, [mailto:jbaumgardner@missouriwestern.edu]<br />
<br />
• Tom Crowley, Senior Biochemisty/Molecular Biology Major, [mailto:stc8033@missouriwestern.edu]<br />
<br />
• Lane H. Heard, Central High School graduate, [mailto:axenmoon@hotmail.com]<br />
<br />
• Nickolaus Morton, Junior Chemistry Major, [mailto:nmorton@missouriwestern.edu]<br />
<br />
• Michelle Ritter, Junior Mathematics Major, [mailto:mrr5418@missouriwestern.edu]<br />
<br />
• Jessica Treece, Junior Biology Major (Health Sciences), [mailto:jtreece@missouriwestern.edu]<br />
<br />
• Matthew Unzicker, Senior Biochemistry/Molecular Biology Major, [mailto:mru8487@missouriwestern.edu]<br />
<br />
• Amanda Valencia, Senior Biochem/Molecular Biology Major, [mailto:avalencia@missouriwestern.edu]<br />
<br />
== '''Faculty''' ==<br />
[[Image:Logo.gif]]<br />
<br />
• A. Malcolm Campbell [http://www.bio.davidson.edu/people/macampbell/macampbell.html], Professor, Department of Biology, [mailto:macampbell@davidson.edu]<br />
<br />
• [https://2007.igem.org/User:Kahaynes Karmella Haynes], Visiting Assistant Professor, Department of Biology, [mailto:kahaynes@davidson.edu]<br />
<br />
• [http://www.davidson.edu/math/heyer/ Laurie Heyer], Associate Professor, Department of Mathematics, [mailto:laheyer@davidson.edu]<br />
<br />
Shipping Address: Malcolm Campbell, Biology Dept. Davidson College, 209 Ridge Road, Davidson, NC 28036 [(704) 894-2692]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
• Todd Eckdahl [http://staff.missouriwestern.edu/~eckdahl/], Professor, Department of Biology, [mailto:eckdahl@missouriwestern.edu]<br />
<br />
• Jeff Poet [http://staff.missouriwestern.edu/~poet/], Assistant Professor, Department of Computer Science, Mathematics, and Physics, [mailto:poet@missouriwestern.edu]<br />
<br />
Shipping Address: Todd Eckdahl, Biology Department, Missouri Western State University, 4525 Downs Drive, Saint Joseph, MO, 64507 [(816) 271-5873]<br />
<br />
== '''Project Overview'''==<br />
<br />
<font color="blue">Hamiltonian Path Problem</font color> <br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/hix DNA recombination mechanism which exists in nature in Salmonella as standard biobricks for use in E. coli. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we continue our collaboration and our efforts to manipulate E. coli into mathematics problem solvers as we refine our efforts with the hin/hix mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adelman in 1994 (citation below) where a unique Hamiltonian path was found in vitro for a particular directed graph on seven nodes. We propose to make progress toward solving the particular problem in vivo.<br />
<br />
[[Image:AdlemanGraph.jpg|thumb|300px|center| ]] <br />
<br><br />
[[Image:Sample_graph.png|thumb|700px|center|]]<br />
<br />
==[[Splitting Genes for HPP]]==<br />
<br />
==[[Davidson's First HPP Construct]]==<br />
<br />
==[[Missouri Western's First HPP Construct]]==<br />
<br />
==[[Assembly Plan]]==<br />
<br />
[[Image:HPPAssemblyPlan72007.jpg|thumb|600px|center| ]]<br />
<br />
==[[Traveling Salesperson Problem?]]==<br />
<br />
==[[Synthesis of First Bacterial Computer]]==<br />
== '''Resources / Citations'''==<br />
<br />
[https://2006.igem.org/wiki/index.php/The_What%27s_and_How%27s Davidson's Wet Lab Protocols]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/MWSU_protocols Missouri Western's Wet Lab Protocols]<br />
<br />
[http://gcat.davidson.edu/iGEM07/genesplitter.html Spliting Genes Web Tool]<br />
<br />
[http://www.bio.davidson.edu/courses/Molbio/Protocols/ORIs.html '''Compatibility of Plasmids''']<br />
<br />
[https://2006.igem.org/wiki/index.php/Freezer_Stocks Freezer Stocks - iGEM 2006 Project]<br />
<br />
[http://spreadsheets.google.com/pub?key=pw-NamR_FPJOfhl6mDrkZcw Davidson Freezer Stocks - iGEM 2007 Project]<br />
<br />
#'''DMW Part Numbers for 2007 are BBa_I715000 to BBa_I715999.''' <br />
#[http://partsregistry.org/Help:BioBrick_Part_Names How to Name a New Part]<br />
#[http://partsregistry.org/Add_a_Part_to_the_Registry Entering the Part to the Registry]<br />
#[http://partsregistry.org/Help:Part_Features How to Annotate a Part]<br />
<br />
Cool site for Breakfast<br />
[http://www.cut-the-knot.org/SimpleGames/Flipper.shtml]<br />
<br />
Karen Acker's paper describing GFP and TetA(c) with Hix insertions<br />
[http://www.bio.davidson.edu/Courses/Immunology/Students/spring2006/Acker/Acker_finalpaperGFP.doc]<br />
<br />
Bruce Henschen's paper describing one-time flippable Hix sites<br />
[http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bruce_Finalpaper.doc]<br />
<br />
Intro to Hamiltonian Path Problem and DNA<br />
[http://www.ams.org/featurecolumn/archive/dna-abc2.html]<br />
<br />
Adleman, LM. Molecular Computation of Solutions To Combinatorial Problems. Science. 11 November 1994. Vol. 266. no. 5187, pp. 1021 - 1024<br />
<br />
Sambrook and Russell. 2001. Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratry Press. Cold Spring Harbor, New York pg. 1.145. 2007 June.<br />
<br />
==[[Literature Research]]==</div>Macampbellhttp://2007.igem.org/wiki/index.php/AwardsAwards2007-08-29T20:21:11Z<p>Macampbell: </p>
<hr />
<div>The awards committee needs members and Judges to evaluate the iGEM 2007 projects. Sign<br />
up here if you are interested.<br />
<br />
This year we will be giving awards in categories you will be expected to enter the formal<br />
competition in a category:<br />
<br />
(This list is not final - yet)<br />
<br />
* Health and Medicine<br />
* Energy and the Environment<br />
* Parts and Devices<br />
* Fun and Games<br />
* Sensors<br />
* Computational/Mathematical/Measurement <br />
* Other<br />
<br />
<br />
Awards Committee:<br />
<br />
* Drew Endy - Co-Chair<br />
* Chris Voigt - Co-chair<br />
<br />
Members:<br />
<br />
* Tom Richard - Penn State<br />
* Malcolm Campbell - Davidson College<br />
<br />
Judges:</div>Macampbellhttp://2007.igem.org/wiki/index.php/AwardsAwards2007-08-29T20:20:38Z<p>Macampbell: </p>
<hr />
<div>The awards committee needs members and Judges to evaluate the iGEM 2007 projects. Sign<br />
up here if you are interested.<br />
<br />
This year we will be giving awards in categories you will be expected to enter the formal<br />
competition in a category:<br />
<br />
(This list is not final - yet)<br />
<br />
* Health and Medicine<br />
* Energy and the Environment<br />
* Parts and Devices<br />
* Fun and Games<br />
* Sensors<br />
* Computational/Mathematical<br />
* Other<br />
<br />
<br />
Awards Committee:<br />
<br />
* Drew Endy - Co-Chair<br />
* Chris Voigt - Co-chair<br />
<br />
Members:<br />
<br />
* Tom Richard - Penn State<br />
* Malcolm Campbell - Davidson College<br />
<br />
Judges:</div>Macampbellhttp://2007.igem.org/wiki/index.php/AwardsAwards2007-08-29T20:20:15Z<p>Macampbell: </p>
<hr />
<div>The awards committee needs members and Judges to evaluate the iGEM 2007 projects. Sign<br />
up here if you are interested.<br />
<br />
This year we will be giving awards in categories you will be expected to enter the formal<br />
competition in a category:<br />
<br />
(This list is not final - yet)<br />
<br />
* Health and Medicine<br />
* Energy and the Environment<br />
* Parts and Devices<br />
* Fun and Games<br />
* Sensors<br />
* Computational/Mathematical<br />
* Other<br />
<br />
<br />
Awards Committee:<br />
<br />
* Drew Endy - Co-Chair<br />
* Chris Voigt - Co-chair<br />
<br />
Members:<br />
<br />
* Tom Richard - Penn State<br />
<br />
Judges:</div>Macampbellhttp://2007.igem.org/wiki/index.php/Splitting_Genes_for_HPPSplitting Genes for HPP2007-07-31T00:46:51Z<p>Macampbell: </p>
<hr />
<div>We have selected 4 genes to split. We will use our [http://gcat.davidson.edu/iGEM07/genesplitter.html online gene splitting tool] to choose the PCR primers. Davidson will produce these 4 split genes and test each one. <br />
<br />
# Red Fluorescent Protein<br />
# Kanamycin Nucleotidyltransferase <br />
# Cre Recombinase<br />
# Chloramphenicol Acetyltransferase<br />
<br />
<br />
We will also produce two constructs for tetsing promoters. MWSU will produec (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes. <br />
<br />
Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and lack the backwards promotion we have detected with Part: [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. We will test out the modified promoter [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] which is reported to have good repression and strong induction. We may still introduce the UV5 double mutation to enhance transcription and compare with R0010.<br />
<br />
MWSU is also going to produce backwards LacIq to put upstream of pLac [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. The purpose of this is to have more LacIq in the cytoplasm at all times, regardless of ITPG status. <br />
<br />
MWSU is also testing two of the [http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bio372.html one-time-flippable HixC sites] produced by Bruce spring 2007. <br />
<br />
MWSU is going to produce a new Hin-LVA expression plasmid that has ColE1 ori and uses Tet as the selectable marker. <br />
<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br><br />
----<br />
'''DsRed - Red Fluorescent Protein'''<br />
<br />
We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 ''Discosoma sp.'']), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.<br />
<br />
Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.<br />
<center><br />
[[Image:Rfp hix insertion point.jpg]]<br />
</center><br />
<br><br />
----<br />
<br />
'''Kanamycin Nucleotidyltransferase'''<br />
<br><br />
[[Image:Kanamycin.png|300px|center|]]<br />
<br><br />
One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA.[http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1] <br />
Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimmer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that: <br />
<br />
-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]<br />
<br />
-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-We did not consider any Amino Acids near the N or C terminus <br />
<br />
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)<br />
<br />
<center><br />
[[Image:KNTase hix cut.png]]<br />
<br />
The yellow bands at the top and bottom of the molecule denotes hix site insertion<br />
</center><br />
<br />
We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.<br />
<br><br />
----<br />
<br />
'''Chloramphenicol Acetyltransferase'''<br />
<br><br />
[[Image:800px-Chloramphenicol-2D-skeletal.png|300px|center|]]<br />
<br />
<br />
[http://partsregistry.org/Part:BBa_P1004 Chloramphenicol Acetyltransferase] was one of the genes we chose as a node for our Hamiltonian Path. It is a bacterial gene that neutralizes the effect of an antibiotic, Chloramphenicol, by transferring acetyl groups to Chloramphenicol and changes its shape into a harmless form. <br />
<br />
The specific Chloramphenicol Acetyltransferase gene we are using comes from the plasmid PSV2CAT whose original source is an ''E. coli'' transposable element Tn9 (Sambrook, 2001) Its PDB ID# is [http://www.pdb.org/pdb/explore/explore.do?structureId=1PD5 1PD5]. <br />
<br />
I have chosen to insert my hixC site between amino acid 52 and 53. I chose this point because it is away from the active site of the protein, the point that contains the catalytic binding sites and allow the recognition and binding of the substrate. It is important for the insertion point to be away from the active site because we do not want the overall function and structure of the protein to be destroyed in the process of splitting. We want to split at a point where the two halves of the protein cannot work as single units, but once a hixC site has been inserted, and the two halves are brought back together, the protein displays its original function.<br />
<center><br />
[[Image: 1pd5.jpg]]<br />
<br />
<br />
The structure of a type I Chloramphenicol Acetyltransferase used in the BioBrick Registry. <br />
<br />
<br />
[[Image: type 3.jpg]]<br />
<br />
The structure of a type III Chloramphenicol Acetyltransferase. <br />
</center><br />
<br />
This structure shows the active sites of the protein. Although the two proteins are of different types and the amino acid sequence are different, the structures are similar and we can therefore see that the point where we have decided to split is “safe”. The exact points that I have decided to cut is away from the active site. <br />
<br />
<br />
<br />
<br><br />
----<br />
<br />
'''Cre Recombinase'''<br />
<br />
We will also attempt to insert a hixC site into the Cre Recombinase gene. Cre Recombinase binds as a dimer to a specific sequence of DNA called a ''loxP'' site. If two ''loxP'' sites are facing in opposite orientations, then Cre Recombinase will flip the section of DNA in between. If two ''loxP'' sites are facing in the same orientation, then Cre Recombinase will excise the DNA in between, creating a new plasmid. <br />
<br />
***View the [[Cre Reporter Construct]] to see how we will detect the presence of Cre Recombinase in our HPP computer.<br />
<br />
In researching Cre Recombinase, we found that the gene had already been split by another lab. [http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT] In the study done by Casanova et al, two independent but overlapping sections of the Cre Recombinase gene were placed in separate locations along an E. Coli chromosome. When translated, the two overlapping halves of the Cre Recombinase protein bound together and formed a functional Cre Recombinase protein. <br />
<br />
We believe that in order for the split protein to be functional, the overlapping section of the bound protein at the split site could not significantly hinder the protein’s ability to bind to ''loxP'' sites and recombine DNA segments. With that in mind, we investigated the same region split by Casanova et al. as the prime candidate for the insertion of a hixC site. <br />
<br />
The site that was eventually chosen reflects both the protein structure shown to the right and the previous research done in the Casanova lab. We believe that amino acids 190-191 along the Cre Recombinase protein are unlikely to play a significant role in the functioning of the protein, thus we decided on this location for the insertion of our hixC site.<br />
<br />
Figure 1, below, shows a monomer of Cre Recombinase bound to a DNA strand that it is about the cut. Our split site is highlighted in yellow and can be seen far away from the active site of the molecule. <br />
<br />
Figure 2, below, shows two dimers of Cre Recombinase coming come together to cut DNA at two ''loxP'' sites. The site of our hixC insertion is highlighted in yellow on each molecule and can be seen far away from the active site.<br />
<br />
[[Image:Cre_recombinase_monomer.png|250px]]<br><br />
Figure 1<br><br />
<br />
[[Image:Cre_recombinase_tetramer.png|250px]]<br><br />
Figure 2<br />
<br />
<br />
<br><br />
----<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br></div>Macampbellhttp://2007.igem.org/wiki/index.php/File:800px-Chloramphenicol-2D-skeletal.pngFile:800px-Chloramphenicol-2D-skeletal.png2007-07-31T00:46:06Z<p>Macampbell: </p>
<hr />
<div></div>Macampbellhttp://2007.igem.org/wiki/index.php/File:Kanamycin.jpgFile:Kanamycin.jpg2007-07-06T14:30:33Z<p>Macampbell: </p>
<hr />
<div></div>Macampbellhttp://2007.igem.org/wiki/index.php/Splitting_Genes_for_HPPSplitting Genes for HPP2007-07-06T14:27:32Z<p>Macampbell: </p>
<hr />
<div>We have selected 4 genes to split. We will use our [http://gcat.davidson.edu/iGEM07/genesplitter.html online gene splitting tool] to choose the PCR primers. Davidson will produce these 4 split genes and test each one. <br />
<br />
# Red Fluorescent Protein<br />
# Kanamycin Nucleotidyltransferase <br />
# Cre Recombinase<br />
# Chloramphenicol Acetyltransferase<br />
<br />
<br />
We will also produce two constructs for tetsing promoters. MWSU will produec (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes. <br />
<br />
Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and lack the backwards promotion we have detected with Part: [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. We will test out the modified promoter [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] which is reported to have good repression and strong induction. We may still introduce the UV5 double mutation to enhance transcription and compare with R0010.<br />
<br />
MWSU is also going to produce backwards LacIq to put upstream of pLac [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. The purpose of this is to have more LacIq in the cytoplasm at all times, regardless of ITPG status. <br />
<br />
MWSU is also testing two of the [http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bio372.html one-time-flippable HixC sites] produced by Bruce spring 2007. <br />
<br />
MWSU is going to produce a new Hin-LVA expression plasmid that has ColE1 ori and uses Tet as the selectable marker. <br />
<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br><br />
----<br />
'''DsRed - Red Fluorescent Protein'''<br />
<br />
We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 ''Discosoma sp.'']), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.<br />
<br />
Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.<br />
<center><br />
[[Image:Rfp hix insertion point.jpg]]<br />
</center><br />
<br><br />
----<br />
<br />
'''Kanamycin Nucleotidyltransferase'''<br />
<br><br />
[[Image:Kanamycin.png|300px|center|]]<br />
<br><br />
One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA.[http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1] <br />
Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimmer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that: <br />
<br />
-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]<br />
<br />
-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-We did not consider any Amino Acids near the N or C terminus <br />
<br />
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)<br />
<br />
<center><br />
[[Image:KNTase hix cut.png]]<br />
<br />
The yellow bands at the top and bottom of the molecule denotes hix site insertion<br />
</center><br />
<br />
We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.<br />
<br><br />
----<br />
<br />
'''Chloramphenicol Acetyltransferase'''<br />
<br><br />
[[Image:Tetracycline.png|300px|center|]]<br />
<br />
<br />
[http://partsregistry.org/Part:BBa_P1004 Chloramphenicol Acetyltransferase] was one of the genes we chose as a node for our Hamiltonian Path. It is a bacterial gene that neutralizes the effect of an antibiotic, Chloramphenicol, by transferring acetyl groups to Chloramphenicol and changes its shape into a harmless form. <br />
<br />
The specific Chloramphenicol Acetyltransferase gene we are using comes from the plasmid PSV2CAT whose original source is an ''E. coli'' transposable element Tn9 (Sambrook, 2001) Its PDB ID# is [http://www.pdb.org/pdb/explore/explore.do?structureId=1PD5 1PD5]. <br />
<br />
I have chosen to insert my hixC site between amino acid 52 and 53. I chose this point because it is away from the active site of the protein, the point that contains the catalytic binding sites and allow the recognition and binding of the substrate. It is important for the insertion point to be away from the active site because we do not want the overall function and structure of the protein to be destroyed in the process of splitting. We want to split at a point where the two halves of the protein cannot work as single units, but once a hixC site has been inserted, and the two halves are brought back together, the protein displays its original function.<br />
<center><br />
[[Image: 1pd5.jpg]]<br />
<br />
<br />
The structure of a type I Chloramphenicol Acetyltransferase used in the BioBrick Registry. <br />
<br />
<br />
[[Image: type 3.jpg]]<br />
<br />
The structure of a type III Chloramphenicol Acetyltransferase. <br />
</center><br />
<br />
This structure shows the active sites of the protein. Although the two proteins are of different types and the amino acid sequence are different, the structures are similar and we can therefore see that the point where we have decided to split is “safe”. The exact points that I have decided to cut is away from the active site. <br />
<br />
<br />
<br />
<br><br />
----<br />
<br />
'''Cre Recombinase'''<br />
<br />
We will also attempt to insert a hixC site into the Cre Recombinase gene. Cre Recombinase binds as a dimer to a specific sequence of DNA called a ''loxP'' site. If two ''loxP'' sites are facing in opposite orientations, then Cre Recombinase will flip the section of DNA in between. If two ''loxP'' sites are facing in the same orientation, then Cre Recombinase will excise the DNA in between, creating a new plasmid. <br />
<br />
***View the [[Cre Reporter Construct]] to see how we will detect the presence of Cre Recombinase in our HPP computer.<br />
<br />
In researching Cre Recombinase, we found that the gene had already been split by another lab. [http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT] In the study done by Casanova et al, two independent but overlapping sections of the Cre Recombinase gene were placed in separate locations along an E. Coli chromosome. When translated, the two overlapping halves of the Cre Recombinase protein bound together and formed a functional Cre Recombinase protein. <br />
<br />
We believe that in order for the split protein to be functional, the overlapping section of the bound protein at the split site could not significantly hinder the protein’s ability to bind to ''loxP'' sites and recombine DNA segments. With that in mind, we investigated the same region split by Casanova et al. as the prime candidate for the insertion of a hixC site. <br />
<br />
The site that was eventually chosen reflects both the protein structure shown to the right and the previous research done in the Casanova lab. We believe that amino acids 190-191 along the Cre Recombinase protein are unlikely to play a significant role in the functioning of the protein, thus we decided on this location for the insertion of our hixC site.<br />
<br />
Figure 1, below, shows a monomer of Cre Recombinase bound to a DNA strand that it is about the cut. Our split site is highlighted in yellow and can be seen far away from the active site of the molecule. <br />
<br />
Figure 2, below, shows two dimers of Cre Recombinase coming come together to cut DNA at two ''loxP'' sites. The site of our hixC insertion is highlighted in yellow on each molecule and can be seen far away from the active site.<br />
<br />
[[Image:Cre_recombinase_monomer.png|250px]]<br><br />
Figure 1<br><br />
<br />
[[Image:Cre_recombinase_tetramer.png|250px]]<br><br />
Figure 2<br />
<br />
<br />
<br><br />
----<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br></div>Macampbellhttp://2007.igem.org/wiki/index.php/File:Tetracycline.pngFile:Tetracycline.png2007-07-06T14:24:11Z<p>Macampbell: </p>
<hr />
<div></div>Macampbellhttp://2007.igem.org/wiki/index.php/File:Kanamycin.pngFile:Kanamycin.png2007-07-06T14:23:37Z<p>Macampbell: </p>
<hr />
<div></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-07-05T18:55:28Z<p>Macampbell: /* Davidson & Missouri Western Team Logos, iGEM2006 */</p>
<hr />
<div>===Davidson & Missouri Western Team Logos, iGEM2006=== <br><br />
DMW.jpg<br />
<br />
[[Image:DMW.jpg|thumb|300px|center| ]]<br />
<br><br />
[[Image:Ihop.PNG|thumb|300px|center| ]]<br />
<br><br />
<br />
----<br />
<br />
== '''Team Meeting Notes''' ==<br />
<br />
Western Meeting Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/WesternMeetingNotes]<br />
<br />
== '''Math Related Notes''' ==<br />
<br />
Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/MathMeetingNotes]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/FalsePositiveProgram False Positives]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/ProjectsCompleted Projects Completed]<br />
<br />
== '''Students''' ==<br />
<br />
[[Image:Logo.gif]]<br />
<br />
• Will DeLoache, Junior Biology Major, [mailto:wideloache@davidson.edu]<br />
<br />
• Oyinade Adefuye, Senior Biology Major, [mailto:oyinadeadefuye@yahoo.com]<br />
<br />
• Jim Dickson, Junior Math and Economics Major, [mailto:jidickson@davidson.edu]<br />
<br />
• Amber Shoecraft, Math Major, [mailto:amshoecraft@davidson.edu]<br />
<br />
• Andrew Martens, Senior Biology Major, [mailto:anmartens@davidson.edu]<br />
<br />
• Michael Waters, Sophomore Biology Major, [mailto:miwaters@davidson.edu]<br />
<br><br />
[[Image:Team1.jpg|200px|]] [[Image:Team_graph.jpg|200px|]]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
<br />
• Jordan Baumgardner, Junior Biology, Biochemistry/Molecular Biology Major, [mailto:jbaumgardner@missouriwestern.edu]<br />
<br />
• Tom Crowley, Senior Biochemisty/Molecular Biology Major, [mailto:stc8033@missouriwestern.edu]<br />
<br />
• Lane H. Heard, Central High School graduate, [mailto:axenmoon@hotmail.com]<br />
<br />
• Nickolaus Morton, Junior Chemistry Major, [mailto:nmorton@missouriwestern.edu]<br />
<br />
• Michelle Ritter, Junior Mathematics Major, [mailto:mrr5418@missouriwestern.edu]<br />
<br />
• Jessica Treece, Junior Biology Major (Health Sciences), [mailto:jtreece@missouriwestern.edu]<br />
<br />
• Matthew Unzicker, Senior Biochemistry/Molecular Biology Major, [mailto:mru8487@missouriwestern.edu]<br />
<br />
• Amanda Valencia, Senior Biochem/Molecular Biology Major, [mailto:avalencia@missouriwestern.edu]<br />
<br />
== '''Faculty''' ==<br />
[[Image:Logo.gif]]<br />
<br />
• A. Malcolm Campbell [http://www.bio.davidson.edu/people/macampbell/macampbell.html], Professor, Department of Biology, [mailto:macampbell@davidson.edu]<br />
<br />
• [http://www.bio.davidson.edu/people/kahaynes/kahaynes.html Karmella Haynes], Visiting Assistant Professor, Department of Biology, [mailto:kahaynes@davidson.edu]<br />
<br />
• Laurie Heyer [http://www.davidson.edu/math/heyer/], Associate Professor, Department of Mathematics, [mailto:laheyer@davidson.edu]<br />
<br />
Shipping Address: Malcolm Campbell, Biology Dept. Davidson College, 209 Ridge Road, Davidson, NC 28036 [(704) 894-2692]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
• Todd Eckdahl [http://staff.missouriwestern.edu/~eckdahl/], Professor, Department of Biology, [mailto:eckdahl@missouriwestern.edu]<br />
<br />
• Jeff Poet [http://staff.missouriwestern.edu/~poet/], Assistant Professor, Department of Computer Science, Mathematics, and Physics, [mailto:poet@missouriwestern.edu]<br />
<br />
Shipping Address: Todd Eckdahl, Biology Department, Missouri Western State University, 4525 Downs Drive, Saint Joseph, MO, 64507 [(816) 271-5873]<br />
<br />
== '''Project Overview'''==<br />
<br />
<font color="blue">Hamiltonian Path Problem</font color> <br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/hix DNA recombination mechanism which exists in nature in Salmonella as standard biobricks for use in E. coli. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we continue our collaboration and our efforts to manipulate E. coli into mathematics problem solvers as we refine our efforts with the hin/hix mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adelman in 1994 (citation below) where a unique Hamiltonian path was found in vitro for a particular directed graph on seven nodes. We propose to make progress toward solving the particular problem in vivo.<br />
<br />
[[Image:AdlemanGraph.jpg|thumb|300px|center| ]] <br />
<br><br />
[[Image:Sample_graph.png|thumb|700px|center|]]<br />
<br />
==[[Splitting Genes for HPP]]==<br />
<br />
==[[Davidson's First HPP Construct]]==<br />
<br />
==[[Missouri Western's First HPP Construct]]==<br />
<br />
==[[Traveling Salesperson Problem?]]==<br />
<br />
== '''Resources / Citations'''==<br />
<br />
[https://2006.igem.org/wiki/index.php/The_What%27s_and_How%27s Davidson's Wet Lab Protocols]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/MWSU_protocols Missouri Western's Wet Lab Protocols]<br />
<br />
[http://gcat.davidson.edu/iGEM07/genesplitter.html Spliting Genes Web Tool]<br />
<br />
[http://www.bio.davidson.edu/courses/Molbio/Protocols/ORIs.html '''Compatibility of Plasmids''']<br />
<br />
[https://2006.igem.org/wiki/index.php/Freezer_Stocks Freezer Stocks - iGEM 2006 Project]<br />
<br />
#'''DMW Part Numbers for 2007 are BBa_I715000 to BBa_I715999.''' <br />
#[http://partsregistry.org/Help:BioBrick_Part_Names How to Name a New Part]<br />
#[http://partsregistry.org/Add_a_Part_to_the_Registry Entering the Part to the Registry]<br />
#[http://partsregistry.org/Help:Part_Features How to Annotate a Part]<br />
<br />
Cool site for Breakfast<br />
[http://www.cut-the-knot.org/SimpleGames/Flipper.shtml]<br />
<br />
Karen Acker's paper describing GFP and TetA(c) with Hix insertions<br />
[http://www.bio.davidson.edu/Courses/Immunology/Students/spring2006/Acker/Acker_finalpaperGFP.doc]<br />
<br />
Bruce Henschen's paper describing one-time flippable Hix sites<br />
[http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bruce_Finalpaper.doc]<br />
<br />
Intro to Hamiltonian Path Problem and DNA<br />
[http://www.ams.org/featurecolumn/archive/dna-abc2.html]<br />
<br />
Adleman, LM. Molecular Computation of Solutions To Combinatorial Problems. Science. 11 November 1994. Vol. 266. no. 5187, pp. 1021 - 1024<br />
<br />
Sambrook and Russell. 2001. Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratry Press. Cold Spring Harbor, New York pg. 1.145. 2007 June.<br />
<br />
==[[Literature Research]]==</div>Macampbellhttp://2007.igem.org/wiki/index.php/File:DMW.jpgFile:DMW.jpg2007-07-05T18:54:47Z<p>Macampbell: simple logo for DMW</p>
<hr />
<div>simple logo for DMW</div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-06-29T19:22:34Z<p>Macampbell: /* '''Students''' */</p>
<hr />
<div>===Davidson & Missouri Western Team Logos, iGEM2006=== <br><br />
[[Image:EHOP.gif|thumb|300px|center| ]]<br />
<br><br />
[[Image:Ihop.PNG|thumb|300px|center| ]]<br />
<br><br />
<br />
----<br />
== '''Team Meeting Notes''' ==<br />
<br />
Western Meeting Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/WesternMeetingNotes]<br />
<br />
== '''Math Related Notes''' ==<br />
<br />
Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/MathMeetingNotes]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/FalsePositiveProgram False Positives]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/ProjectsCompleted Projects Completed]<br />
<br />
== '''Students''' ==<br />
<br />
[[Image:Logo.gif]]<br />
<br />
• Will DeLoache, Junior Biology Major, [mailto:wideloache@davidson.edu]<br />
<br />
• Oyinade Adefuye, Senior Biology Major, [mailto:oyinadeadefuye@yahoo.com]<br />
<br />
• Jim Dickson, Junior Math and Economics Major, [mailto:jidickson@davidson.edu]<br />
<br />
• Amber Shoecraft, Math Major, [mailto:amshoecraft@davidson.edu]<br />
<br />
• Andrew Martens, Senior Biology Major, [mailto:anmartens@davidson.edu]<br />
<br />
• Michael Waters, Sophomore Biology Major, [mailto:miwaters@davidson.edu]<br />
<br><br />
[[Image:Team1.jpg|200px|]] [[Image:Team_graph.jpg|200px|]]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
<br />
• Jordan Baumgardner, Junior Biology, Biochemistry/Molecular Biology Major, [mailto:jbaumgardner@missouriwestern.edu]<br />
<br />
• Tom Crowley, Senior Biochemisty/Molecular Biology Major, [mailto:stc8033@missouriwestern.edu]<br />
<br />
• Lane H. Heard, Central High School graduate, [mailto:axenmoon@hotmail.com]<br />
<br />
• Nickolaus Morton, Junior Chemistry Major, [mailto:nmorton@missouriwestern.edu]<br />
<br />
• Michelle Ritter, Junior Mathematics Major, [mailto:mrr5418@missouriwestern.edu]<br />
<br />
• Jessica Treece, Junior Biology Major (Health Sciences), [mailto:jtreece@missouriwestern.edu]<br />
<br />
• Matthew Unzicker, Senior Biochemistry/Molecular Biology Major, [mailto:mru8487@missouriwestern.edu]<br />
<br />
• Amanda Valencia, Senior Biochem/Molecular Biology Major, [mailto:avalencia@missouriwestern.edu]<br />
<br />
== '''Faculty''' ==<br />
[[Image:Logo.gif]]<br />
<br />
• A. Malcolm Campbell [http://www.bio.davidson.edu/people/macampbell/macampbell.html], Professor, Department of Biology, [mailto:macampbell@davidson.edu]<br />
<br />
• [http://www.bio.davidson.edu/people/kahaynes/kahaynes.html Karmella Haynes], Visiting Assistant Professor, Department of Biology, [mailto:kahaynes@davidson.edu]<br />
<br />
• Laurie Heyer [http://www.davidson.edu/math/heyer/], Associate Professor, Department of Mathematics, [mailto:laheyer@davidson.edu]<br />
<br />
Shipping Address: Malcolm Campbell, Biology Dept. Davidson College, 209 Ridge Road, Davidson, NC 28036 [(704) 894-2692]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
• Todd Eckdahl [http://staff.missouriwestern.edu/~eckdahl/], Professor, Department of Biology, [mailto:eckdahl@missouriwestern.edu]<br />
<br />
• Jeff Poet [http://staff.missouriwestern.edu/~poet/], Assistant Professor, Department of Computer Science, Mathematics, and Physics, [mailto:poet@missouriwestern.edu]<br />
<br />
Shipping Address: Todd Eckdahl, Biology Department, Missouri Western State University, 4525 Downs Drive, Saint Joseph, MO, 64507 [(816) 271-5873]<br />
<br />
== '''Project Overview'''==<br />
<br />
<font color="blue">Hamiltonian Path Problem</font color> <br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/hix DNA recombination mechanism which exists in nature in Salmonella as standard biobricks for use in E. coli. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we continue our collaboration and our efforts to manipulate E. coli into mathematics problem solvers as we refine our efforts with the hin/hix mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adelman in 1994 (citation below) where a unique Hamiltonian path was found in vitro for a particular directed graph on seven nodes. We propose to make progress toward solving the particular problem in vivo.<br />
<br />
[[Image:AdelmanGraph.JPG|thumb|300px|center| ]] <br />
<br><br />
[[Image:Sample_graph.png|thumb|700px|center|]]<br />
<br />
==[[Splitting Genes for HPP]]==<br />
<br />
==[[Davidson's First HPP Construct]]==<br />
<br />
==[[Missouri Western's First HPP Construct]]==<br />
<br />
==[[Traveling Salesperson Problem?]]==<br />
<br />
== '''Resources / Citations'''==<br />
<br />
[https://2006.igem.org/wiki/index.php/The_What%27s_and_How%27s Davidson's Wet Lab Protocols]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/MWSU_protocols Missouri Western's Wet Lab Protocols]<br />
<br />
[http://gcat.davidson.edu/iGEM07/genesplitter.html Spliting Genes Web Tool]<br />
<br />
[http://www.bio.davidson.edu/courses/Molbio/Protocols/ORIs.html '''Compatibility of Plasmids''']<br />
<br />
#'''DMW Part Numbers for 2007 are BBa_I715000 to BBa_I715999.''' <br />
#[http://partsregistry.org/Help:BioBrick_Part_Names How to Name a New Part]<br />
#[http://partsregistry.org/Add_a_Part_to_the_Registry Entering the Part to the Registry]<br />
#[http://partsregistry.org/Help:Part_Features How to Annotate a Part]<br />
<br />
Cool site for Breakfast<br />
[http://www.cut-the-knot.org/SimpleGames/Flipper.shtml]<br />
<br />
Karen Acker's paper describing GFP and TetA(c) with Hix insertions<br />
[http://www.bio.davidson.edu/Courses/Immunology/Students/spring2006/Acker/Acker_finalpaperGFP.doc]<br />
<br />
Bruce Henschen's paper describing one-time flippable Hix sites<br />
[http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bruce_Finalpaper.doc]<br />
<br />
Intro to Hamiltonian Path Problem and DNA<br />
[http://www.ams.org/featurecolumn/archive/dna-abc2.html]<br />
<br />
Adleman, LM. Molecular Computation of Solutions To Combinatorial Problems. Science. 11 November 1994. Vol. 266. no. 5187, pp. 1021 - 1024<br />
<br />
Sambrook and Russell. 2001. Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratry Press. Cold Spring Harbor, New York pg. 1.145. 2007 June.<br />
<br />
==[[Literature Research]]==</div>Macampbellhttp://2007.igem.org/wiki/index.php/File:Team_graph.jpgFile:Team graph.jpg2007-06-29T19:02:06Z<p>Macampbell: graph in humans</p>
<hr />
<div>graph in humans</div>Macampbellhttp://2007.igem.org/wiki/index.php/File:Team1.jpgFile:Team1.jpg2007-06-29T19:01:25Z<p>Macampbell: students and faculty</p>
<hr />
<div>students and faculty</div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-06-21T12:09:04Z<p>Macampbell: /* '''Students''' */</p>
<hr />
<div>===Davidson & Missouri Western Team Logos, iGEM2006=== <br><br />
[[Image:EHOP.gif|thumb|300px|center| ]]<br />
<br><br />
[[Image:Ihop.PNG|thumb|300px|center| ]]<br />
<br><br />
<br />
----<br />
== '''Team Meeting Notes''' ==<br />
<br />
Western Meeting Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/WesternMeetingNotes]<br />
<br />
== '''Math Related Notes''' ==<br />
<br />
Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/MathMeetingNotes]<br />
<br />
== '''Students''' ==<br />
<br />
[[Image:Logo.gif]]<br />
<br />
• Will DeLoache, Junior Biology Major, [mailto:wideloache@davidson.edu]<br />
<br />
• Oyinade Adefuye, Senior Biology Major, [mailto:oyinadeadefuye@yahoo.com]<br />
<br />
• Jim Dickson, Junior Math and Economics Major, [mailto:jidickson@davidson.edu]<br />
<br />
• Amber Shoecraft, Math Major, [mailto:ashoecraft@davidson.edu]<br />
<br />
• Andrew Martens, Senior Biology Major, [mailto:anmartens@davidson.edu]<br />
<br />
• Michael Waters, Sophomore Biology Major, [mailto:miwaters@davidson.edu]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
<br />
• Jordan Baumgardner, Junior Biology, Biochemistry/Molecular Biology Major, [mailto:jbaumgardner@missouriwestern.edu]<br />
<br />
• Ryan Chilcoat, Junior Biology Major (Health Sciences), [mailto:rchilcoat@missouriwestern.edu]<br />
<br />
• Tom Crowley, Senior Biochemisty/Molecular Biology Major, [mailto:stc8033@missouriwestern.edu]<br />
<br />
• Lane H. Heard, Central High School graduate, [mailto:axenmoon@hotmail.com]<br />
<br />
• Nickolaus Morton, Junior Chemistry Major, [mailto:nmorton@missouriwestern.edu]<br />
<br />
• Michelle Ritter, Junior Mathematics Major, [mailto:mrr5418@missouriwestern.edu]<br />
<br />
• Jessica Treece, Junior Biology Major (Health Sciences), [mailto:jtreece@missouriwestern.edu]<br />
<br />
• Matthew Unzicker, Senior Biochemistry/Molecular Biology Major, [mailto:mru8487@missouriwestern.edu]<br />
<br />
• Amanda Valencia, Senior Biochem/Molecular Biology Major, [mailto:avalencia@missouriwestern.edu]<br />
<br />
== '''Faculty''' ==<br />
[[Image:Logo.gif]]<br />
<br />
• A. Malcolm Campbell [http://www.bio.davidson.edu/people/macampbell/macampbell.html], Professor, Department of Biology, [mailto:macampbell@davidson.edu]<br />
<br />
• [http://www.bio.davidson.edu/people/kahaynes/kahaynes.html Karmella Haynes], Visiting Assistant Professor, Department of Biology, [mailto:kahaynes@davidson.edu]<br />
<br />
• Laurie Heyer [http://www.davidson.edu/math/heyer/], Associate Professor, Department of Mathematics, [mailto:laheyer@davidson.edu]<br />
<br />
Shipping Address: Malcolm Campbell, Biology Dept. Davidson College, 209 Ridge Road, Davidson, NC 28036 [(704) 894-2692]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
• Todd Eckdahl [http://staff.missouriwestern.edu/~eckdahl/], Professor, Department of Biology, [mailto:eckdahl@missouriwestern.edu]<br />
<br />
• Jeff Poet [http://staff.missouriwestern.edu/~poet/], Assistant Professor, Department of Computer Science, Mathematics, and Physics, [mailto:poet@missouriwestern.edu]<br />
<br />
Shipping Address: Todd Eckdahl, Biology Department, Missouri Western State University, 4525 Downs Drive, Saint Joseph, MO, 64507 [(816) 271-5873]<br />
<br />
== '''Project Overview'''==<br />
<br />
<font color="blue">Hamiltonian Path Problem</font color> <br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/hix DNA recombination mechanism which exists in nature in Salmonella as standard biobricks for use in E. coli. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we continue our collaboration and our efforts to manipulate E. coli into mathematics problem solvers as we refine our efforts with the hin/hix mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adelman in 1994 (citation below) where a unique Hamiltonian path was found in vitro for a particular directed graph on seven nodes. We propose to make progress toward solving the particular problem in vivo.<br />
<br />
[[Image:AdelmanGraph.JPG|thumb|300px|center| ]] <br />
<br><br />
[[Image:Sample_graph.png|thumb|700px|center|]]<br />
<br />
==[[Splitting Genes for HPP]]==<br />
<br />
== '''Resources / Citations'''==<br />
<br />
[https://2006.igem.org/wiki/index.php/The_What%27s_and_How%27s Davidson's Wet Lab Protocols]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/MWSU_protocols Missouri Western's Wet Lab Protocols]<br />
<br />
[http://gcat.davidson.edu/iGEM07/genesplitter.html Spliting Genes Web Tool]<br />
<br />
[http://www.bio.davidson.edu/courses/Molbio/Protocols/ORIs.html '''Compatibility of Plasmids''']<br />
<br />
#'''DMW Part Numbers for 2007 are BBa_I715000 to BBa_I715999.''' <br />
#[http://partsregistry.org/Help:BioBrick_Part_Names How to Name a New Part]<br />
#[http://partsregistry.org/Add_a_Part_to_the_Registry Entering the Part to the Registry]<br />
#[http://partsregistry.org/Help:Part_Features How to Annotate a Part]<br />
<br />
Cool site for Breakfast<br />
[http://www.cut-the-knot.org/SimpleGames/Flipper.shtml]<br />
<br />
Karen Acker's paper describing GFP and TetA(c) with Hix insertions<br />
[http://www.bio.davidson.edu/Courses/Immunology/Students/spring2006/Acker/Acker_finalpaperGFP.doc]<br />
<br />
Bruce Henschen's paper describing one-time flippable Hix sites<br />
[http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bruce_Finalpaper.doc]<br />
<br />
Intro to Hamiltonian Path Problem and DNA<br />
[http://www.ams.org/featurecolumn/archive/dna-abc2.html]<br />
<br />
Adelman, LM. Molecular Computation of Solutions To Combinatorial Problems. Science. 11 November 1994. Vol. 266. no. 5187, pp. 1021 - 1024<br />
<br />
Ptashne, Mark. A Genetic Switch: Phage Lambda Revisited, Third Edition. New York. Cold Spring Harbor Laboratory Press: 2004.<br />
<br />
Sambrook and Russell. 2001. Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratry Press. Cold Spring Harbor, New York pg. 1.145. 2007 June.<br />
<br />
== '''Literature and Registry Research'''==<br />
<br />
'''Registry Search for Possible Promoters:'''<br />
<br />
BBa_J24669 --- arabinose induced<br />
<br />
BBa_R0082 --- Is upstream of the ompC porin gene<br />
<br />
BBa_R0074 --- Penl regulated<br />
<br />
BBa_I14017 --- P(Rhl)<br />
<br />
BBa_I14018 --- P (Bla) --> amp resistance<br />
<br />
BBa_J3902 --- Pr Fe (Pl + Pll rus operon)<br />
<br />
BBa_R0077 --- CinR --> thought to have own terminator<br />
<br />
BBa_R0078 --- CinR (no RBS)<br />
<br />
BBa_R0062 --- luxR & HSL regulated -- luxpR<br />
<br />
Possibly the use of Constitutive Promoter Family Members to strengthen other promoters.<br />
<br />
'''Literature Search for Polycistronic Genes on Plasmids'''<br />
<br />
Sol Operon<br />
http://www.jstage.jst.go.jp/article/bbb/71/1/58/_pdf<br />
<br />
Transfer (tra) Operon<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1347297&blobtype=pdf<br />
<br />
Oligopeptide Permease (opp)<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1087318&blobtype=pdf</div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-06-21T12:08:17Z<p>Macampbell: /* '''Faculty''' */</p>
<hr />
<div>===Davidson & Missouri Western Team Logos, iGEM2006=== <br><br />
[[Image:EHOP.gif|thumb|300px|center| ]]<br />
<br><br />
[[Image:Ihop.PNG|thumb|300px|center| ]]<br />
<br><br />
<br />
----<br />
== '''Team Meeting Notes''' ==<br />
<br />
Western Meeting Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/WesternMeetingNotes]<br />
<br />
== '''Math Related Notes''' ==<br />
<br />
Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/MathMeetingNotes]<br />
<br />
== '''Students''' ==<br />
<br />
[[Image:Logo.gif]]<br />
<br />
• Will DeLoache, Junior Biology Major, [mailto:wideloache@davidson.edu]<br />
<br />
• Oyinade Adefuye, Senior Biology Major, [mailto:oyinadeadefuye@yahoo.com]<br />
<br />
• Jim Dickson, Junior Math and Economics Major, [mailto:jidickson@davidson.edu]<br />
<br />
• Amber Shoecraft, Math Major, [mailto:ashoecraft@jcsu.edu]<br />
<br />
• Andrew Martens, Senior Biology Major, [mailto:anmartens@davidson.edu]<br />
<br />
• Michael Waters, Sophomore Biology Major, [mailto:miwaters@davidson.edu]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
<br />
• Jordan Baumgardner, Junior Biology, Biochemistry/Molecular Biology Major, [mailto:jbaumgardner@missouriwestern.edu]<br />
<br />
• Ryan Chilcoat, Junior Biology Major (Health Sciences), [mailto:rchilcoat@missouriwestern.edu]<br />
<br />
• Tom Crowley, Senior Biochemisty/Molecular Biology Major, [mailto:stc8033@missouriwestern.edu]<br />
<br />
• Lane H. Heard, Central High School graduate, [mailto:axenmoon@hotmail.com]<br />
<br />
• Nickolaus Morton, Junior Chemistry Major, [mailto:nmorton@missouriwestern.edu]<br />
<br />
• Michelle Ritter, Junior Mathematics Major, [mailto:mrr5418@missouriwestern.edu]<br />
<br />
• Jessica Treece, Junior Biology Major (Health Sciences), [mailto:jtreece@missouriwestern.edu]<br />
<br />
• Matthew Unzicker, Senior Biochemistry/Molecular Biology Major, [mailto:mru8487@missouriwestern.edu]<br />
<br />
• Amanda Valencia, Senior Biochem/Molecular Biology Major, [mailto:avalencia@missouriwestern.edu]<br />
<br />
== '''Faculty''' ==<br />
[[Image:Logo.gif]]<br />
<br />
• A. Malcolm Campbell [http://www.bio.davidson.edu/people/macampbell/macampbell.html], Professor, Department of Biology, [mailto:macampbell@davidson.edu]<br />
<br />
• [http://www.bio.davidson.edu/people/kahaynes/kahaynes.html Karmella Haynes], Visiting Assistant Professor, Department of Biology, [mailto:kahaynes@davidson.edu]<br />
<br />
• Laurie Heyer [http://www.davidson.edu/math/heyer/], Associate Professor, Department of Mathematics, [mailto:laheyer@davidson.edu]<br />
<br />
Shipping Address: Malcolm Campbell, Biology Dept. Davidson College, 209 Ridge Road, Davidson, NC 28036 [(704) 894-2692]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
• Todd Eckdahl [http://staff.missouriwestern.edu/~eckdahl/], Professor, Department of Biology, [mailto:eckdahl@missouriwestern.edu]<br />
<br />
• Jeff Poet [http://staff.missouriwestern.edu/~poet/], Assistant Professor, Department of Computer Science, Mathematics, and Physics, [mailto:poet@missouriwestern.edu]<br />
<br />
Shipping Address: Todd Eckdahl, Biology Department, Missouri Western State University, 4525 Downs Drive, Saint Joseph, MO, 64507 [(816) 271-5873]<br />
<br />
== '''Project Overview'''==<br />
<br />
<font color="blue">Hamiltonian Path Problem</font color> <br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/hix DNA recombination mechanism which exists in nature in Salmonella as standard biobricks for use in E. coli. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we continue our collaboration and our efforts to manipulate E. coli into mathematics problem solvers as we refine our efforts with the hin/hix mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adelman in 1994 (citation below) where a unique Hamiltonian path was found in vitro for a particular directed graph on seven nodes. We propose to make progress toward solving the particular problem in vivo.<br />
<br />
[[Image:AdelmanGraph.JPG|thumb|300px|center| ]] <br />
<br><br />
[[Image:Sample_graph.png|thumb|700px|center|]]<br />
<br />
==[[Splitting Genes for HPP]]==<br />
<br />
== '''Resources / Citations'''==<br />
<br />
[https://2006.igem.org/wiki/index.php/The_What%27s_and_How%27s Davidson's Wet Lab Protocols]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/MWSU_protocols Missouri Western's Wet Lab Protocols]<br />
<br />
[http://gcat.davidson.edu/iGEM07/genesplitter.html Spliting Genes Web Tool]<br />
<br />
[http://www.bio.davidson.edu/courses/Molbio/Protocols/ORIs.html '''Compatibility of Plasmids''']<br />
<br />
#'''DMW Part Numbers for 2007 are BBa_I715000 to BBa_I715999.''' <br />
#[http://partsregistry.org/Help:BioBrick_Part_Names How to Name a New Part]<br />
#[http://partsregistry.org/Add_a_Part_to_the_Registry Entering the Part to the Registry]<br />
#[http://partsregistry.org/Help:Part_Features How to Annotate a Part]<br />
<br />
Cool site for Breakfast<br />
[http://www.cut-the-knot.org/SimpleGames/Flipper.shtml]<br />
<br />
Karen Acker's paper describing GFP and TetA(c) with Hix insertions<br />
[http://www.bio.davidson.edu/Courses/Immunology/Students/spring2006/Acker/Acker_finalpaperGFP.doc]<br />
<br />
Bruce Henschen's paper describing one-time flippable Hix sites<br />
[http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bruce_Finalpaper.doc]<br />
<br />
Intro to Hamiltonian Path Problem and DNA<br />
[http://www.ams.org/featurecolumn/archive/dna-abc2.html]<br />
<br />
Adelman, LM. Molecular Computation of Solutions To Combinatorial Problems. Science. 11 November 1994. Vol. 266. no. 5187, pp. 1021 - 1024<br />
<br />
Ptashne, Mark. A Genetic Switch: Phage Lambda Revisited, Third Edition. New York. Cold Spring Harbor Laboratory Press: 2004.<br />
<br />
Sambrook and Russell. 2001. Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratry Press. Cold Spring Harbor, New York pg. 1.145. 2007 June.<br />
<br />
== '''Literature and Registry Research'''==<br />
<br />
'''Registry Search for Possible Promoters:'''<br />
<br />
BBa_J24669 --- arabinose induced<br />
<br />
BBa_R0082 --- Is upstream of the ompC porin gene<br />
<br />
BBa_R0074 --- Penl regulated<br />
<br />
BBa_I14017 --- P(Rhl)<br />
<br />
BBa_I14018 --- P (Bla) --> amp resistance<br />
<br />
BBa_J3902 --- Pr Fe (Pl + Pll rus operon)<br />
<br />
BBa_R0077 --- CinR --> thought to have own terminator<br />
<br />
BBa_R0078 --- CinR (no RBS)<br />
<br />
BBa_R0062 --- luxR & HSL regulated -- luxpR<br />
<br />
Possibly the use of Constitutive Promoter Family Members to strengthen other promoters.<br />
<br />
'''Literature Search for Polycistronic Genes on Plasmids'''<br />
<br />
Sol Operon<br />
http://www.jstage.jst.go.jp/article/bbb/71/1/58/_pdf<br />
<br />
Transfer (tra) Operon<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1347297&blobtype=pdf<br />
<br />
Oligopeptide Permease (opp)<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1087318&blobtype=pdf</div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-06-20T13:44:11Z<p>Macampbell: /* '''Resources / Citations''' */</p>
<hr />
<div>===Davidson & Missouri Western Team Logos, iGEM2006=== <br><br />
[[Image:EHOP.gif|thumb|300px|center| ]]<br />
<br><br />
[[Image:Ihop.PNG|thumb|300px|center| ]]<br />
<br><br />
<br />
----<br />
== '''Team Meeting Notes''' ==<br />
<br />
Western Meeting Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/WesternMeetingNotes]<br />
<br />
== '''Math Related Notes''' ==<br />
<br />
Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/MathMeetingNotes]<br />
<br />
== '''Students''' ==<br />
<br />
[[Image:Logo.gif]]<br />
<br />
• Will DeLoache, Junior Biology Major, [mailto:wideloache@davidson.edu]<br />
<br />
• Oyinade Adefuye, Senior Biology Major, [mailto:oyinadeadefuye@yahoo.com]<br />
<br />
• Jim Dickson, Junior Math and Economics Major, [mailto:jidickson@davidson.edu]<br />
<br />
• Amber Shoecraft, Math Major, [mailto:ashoecraft@jcsu.edu]<br />
<br />
• Andrew Martens, Senior Biology Major, [mailto:anmartens@davidson.edu]<br />
<br />
• Michael Waters, Sophomore Biology Major, [mailto:miwaters@davidson.edu]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
<br />
• Jordan Baumgardner, Junior Biology, Biochemistry/Molecular Biology Major, [mailto:jbaumgardner@missouriwestern.edu]<br />
<br />
• Ryan Chilcoat, Junior Biology Major (Health Sciences), [mailto:rchilcoat@missouriwestern.edu]<br />
<br />
• Tom Crowley, Senior Biochemisty/Molecular Biology Major, [mailto:stc8033@missouriwestern.edu]<br />
<br />
• Lane H. Heard, Central High School graduate, [mailto:axenmoon@hotmail.com]<br />
<br />
• Nickolaus Morton, Junior Chemistry Major, [mailto:nmorton@missouriwestern.edu]<br />
<br />
• Michelle Ritter, Junior Mathematics Major, [mailto:mrr5418@missouriwestern.edu]<br />
<br />
• Jessica Treece, Junior Biology Major (Health Sciences), [mailto:jtreece@missouriwestern.edu]<br />
<br />
• Matthew Unzicker, Senior Biochemistry/Molecular Biology Major, [mailto:mru8487@missouriwestern.edu]<br />
<br />
• Amanda Valencia, Senior Biochem/Molecular Biology Major, [mailto:avalencia@missouriwestern.edu]<br />
<br />
== '''Faculty''' ==<br />
[[Image:Logo.gif]]<br />
<br />
• Malcolm Campbell [http://www.bio.davidson.edu/people/macampbell/macampbell.html], Professor, Department of Biology, [mailto:macampbell@davidson.edu]<br />
<br />
• [http://www.bio.davidson.edu/people/kahaynes/kahaynes.html Karmella Haynes], Visiting Assistant Professor, Department of Biology, [mailto:kahaynes@davidson.edu]<br />
<br />
• Laurie Heyer [http://www.davidson.edu/math/heyer/], Associate Professor, Department of Mathematics, [mailto:laheyer@davidson.edu]<br />
<br />
Shipping Address: Malcolm Campbell, Biology Dept. Davidson College, 209 Ridge Road, Davidson, NC 28036 [(704) 894-2692]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
• Todd Eckdahl [http://staff.missouriwestern.edu/~eckdahl/], Professor, Department of Biology, [mailto:eckdahl@missouriwestern.edu]<br />
<br />
• Jeff Poet [http://staff.missouriwestern.edu/~poet/], Assistant Professor, Department of Computer Science, Mathematics, and Physics, [mailto:poet@missouriwestern.edu]<br />
<br />
Shipping Address: Todd Eckdahl, Biology Department, Missouri Western State University, 4525 Downs Drive, Saint Joseph, MO, 64507 [(816) 271-5873]<br />
<br />
== '''Project Overview'''==<br />
<br />
<font color="blue">Hamiltonian Path Problem</font color> <br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/hix DNA recombination mechanism which exists in nature in Salmonella as standard biobricks for use in E. coli. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we continue our collaboration and our efforts to manipulate E. coli into mathematics problem solvers as we refine our efforts with the hin/hix mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adelman in 1994 (citation below) where a unique Hamiltonian path was found in vitro for a particular directed graph on seven nodes. We propose to make progress toward solving the particular problem in vivo.<br />
<br />
[[Image:AdelmanGraph.JPG|thumb|300px|center| ]] <br />
<br><br />
[[Image:Sample_graph.png|thumb|700px|center|]]<br />
<br />
==[[Splitting Genes for HPP]]==<br />
<br />
== '''Resources / Citations'''==<br />
<br />
[https://2006.igem.org/wiki/index.php/The_What%27s_and_How%27s Davidson's Wet Lab Protocols]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/MWSU_protocols Missouri Western's Wet Lab Protocols]<br />
<br />
[http://gcat.davidson.edu/iGEM07/genesplitter.html Spliting Genes Web Tool]<br />
<br />
[http://www.bio.davidson.edu/courses/Molbio/Protocols/ORIs.html '''Compatibility of Plasmids''']<br />
<br />
#'''DMW Part Numbers for 2007 are BBa_I715000 to BBa_I715999.''' <br />
#[http://partsregistry.org/Help:BioBrick_Part_Names How to Name a New Part]<br />
#[http://partsregistry.org/Add_a_Part_to_the_Registry Entering the Part to the Registry]<br />
#[http://partsregistry.org/Help:Part_Features How to Annotate a Part]<br />
<br />
Cool site for Breakfast<br />
[http://www.cut-the-knot.org/SimpleGames/Flipper.shtml]<br />
<br />
Karen Acker's paper describing GFP and TetA(c) with Hix insertions<br />
[http://www.bio.davidson.edu/Courses/Immunology/Students/spring2006/Acker/Acker_finalpaperGFP.doc]<br />
<br />
Bruce Henschen's paper describing one-time flippable Hix sites<br />
[http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bruce_Finalpaper.doc]<br />
<br />
Intro to Hamiltonian Path Problem and DNA<br />
[http://www.ams.org/featurecolumn/archive/dna-abc2.html]<br />
<br />
Adelman, LM. Molecular Computation of Solutions To Combinatorial Problems. Science. 11 November 1994. Vol. 266. no. 5187, pp. 1021 - 1024<br />
<br />
Ptashne, Mark. A Genetic Switch: Phage Lambda Revisited, Third Edition. New York. Cold Spring Harbor Laboratory Press: 2004.<br />
<br />
Sambrook and Russell. 2001. Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratry Press. Cold Spring Harbor, New York pg. 1.145. 2007 June.<br />
<br />
== '''Literature and Registry Research'''==<br />
<br />
'''Registry Search for Possible Promoters:'''<br />
<br />
BBa_J24669 --- arabinose induced<br />
<br />
BBa_R0082 --- Is upstream of the ompC porin gene<br />
<br />
BBa_R0074 --- Penl regulated<br />
<br />
BBa_I14017 --- P(Rhl)<br />
<br />
BBa_I14018 --- P (Bla) --> amp resistance<br />
<br />
BBa_J3902 --- Pr Fe (Pl + Pll rus operon)<br />
<br />
BBa_R0077 --- CinR --> thought to have own terminator<br />
<br />
BBa_R0078 --- CinR (no RBS)<br />
<br />
BBa_R0062 --- luxR & HSL regulated -- luxpR<br />
<br />
Possibly the use of Constitutive Promoter Family Members to strengthen other promoters.<br />
<br />
'''Literature Search for Polycistronic Genes on Plasmids'''<br />
<br />
Sol Operon<br />
http://www.jstage.jst.go.jp/article/bbb/71/1/58/_pdf<br />
<br />
Transfer (tra) Operon<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1347297&blobtype=pdf<br />
<br />
Oligopeptide Permease (opp)<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1087318&blobtype=pdf</div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-06-19T13:16:29Z<p>Macampbell: /* '''Resources / Citations''' */</p>
<hr />
<div>===Davidson & Missouri Western Team Logos, iGEM2006=== <br><br />
[[Image:EHOP.gif|thumb|300px|center| ]]<br />
<br><br />
[[Image:Ihop.PNG|thumb|300px|center| ]]<br />
<br><br />
<br />
----<br />
== '''Team Meeting Notes''' ==<br />
<br />
Western Meeting Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/WesternMeetingNotes]<br />
<br />
== '''Math Related Notes''' ==<br />
<br />
Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/MathMeetingNotes]<br />
<br />
== '''Students''' ==<br />
<br />
[[Image:Logo.gif]]<br />
<br />
• Will DeLoache, Junior Biology Major, [mailto:wideloache@davidson.edu]<br />
<br />
• Oyinade Adefuye, Senior Biology Major, [mailto:oyinadeadefuye@yahoo.com]<br />
<br />
• Jim Dickson, Junior Math and Economics Major, [mailto:jidickson@davidson.edu]<br />
<br />
• Amber Shoecraft, Math Major, [mailto:ashoecraft@jcsu.edu]<br />
<br />
• Andrew Martens, Senior Biology Major, [mailto:anmartens@davidson.edu]<br />
<br />
• Michael Waters, Sophomore Biology Major, [mailto:miwaters@davidson.edu]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
<br />
• Jordan Baumgardner, Junior Biology, Biochemistry/Molecular Biology Major, [mailto:jbaumgardner@missouriwestern.edu]<br />
<br />
• Ryan Chilcoat, Junior Biology Major (Health Sciences), [mailto:rchilcoat@missouriwestern.edu]<br />
<br />
• Tom Crowley, Senior Biochemisty/Molecular Biology Major, [mailto:stc8033@missouriwestern.edu]<br />
<br />
• Lane H. Heard, Central High School graduate, [mailto:axenmoon@hotmail.com]<br />
<br />
• Nickolaus Morton, Junior Chemistry Major, [mailto:nmorton@missouriwestern.edu]<br />
<br />
• Michelle Ritter, Junior Mathematics Major, [mailto:mrr5418@missouriwestern.edu]<br />
<br />
• Jessica Treece, Junior Biology Major (Health Sciences), [mailto:jtreece@missouriwestern.edu]<br />
<br />
• Matthew Unzicker, Senior Biochemistry/Molecular Biology Major, [mailto:mru8487@missouriwestern.edu]<br />
<br />
• Amanda Valencia, Senior Biochem/Molecular Biology Major, [mailto:avalencia@missouriwestern.edu]<br />
<br />
== '''Faculty''' ==<br />
[[Image:Logo.gif]]<br />
<br />
• Malcolm Campbell [http://www.bio.davidson.edu/people/macampbell/macampbell.html], Professor, Department of Biology, [mailto:macampbell@davidson.edu]<br />
<br />
• [http://www.bio.davidson.edu/people/kahaynes/kahaynes.html Karmella Haynes], Visiting Assistant Professor, Department of Biology, [mailto:kahaynes@davidson.edu]<br />
<br />
• Laurie Heyer [http://www.davidson.edu/math/heyer/], Associate Professor, Department of Mathematics, [mailto:laheyer@davidson.edu]<br />
<br />
Shipping Address: Malcolm Campbell, Biology Dept. Davidson College, 209 Ridge Road, Davidson, NC 28036 [(704) 894-2692]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
• Todd Eckdahl [http://staff.missouriwestern.edu/~eckdahl/], Professor, Department of Biology, [mailto:eckdahl@missouriwestern.edu]<br />
<br />
• Jeff Poet [http://staff.missouriwestern.edu/~poet/], Assistant Professor, Department of Computer Science, Mathematics, and Physics, [mailto:poet@missouriwestern.edu]<br />
<br />
Shipping Address: Todd Eckdahl, Biology Department, Missouri Western State University, 4525 Downs Drive, Saint Joseph, MO, 64507 [(816) 271-5873]<br />
<br />
== '''Project Overview'''==<br />
<br />
<font color="blue">Hamiltonian Path Problem</font color> <br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/hix DNA recombination mechanism which exists in nature in Salmonella as standard biobricks for use in E. coli. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we continue our collaboration and our efforts to manipulate E. coli into mathematics problem solvers as we refine our efforts with the hin/hix mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adelman in 1994 (citation below) where a unique Hamiltonian path was found in vitro for a particular directed graph on seven nodes. We propose to make progress toward solving the particular problem in vivo.<br />
<br />
[[Image:AdelmanGraph.JPG|thumb|300px|center| ]] <br />
<br><br />
<br />
<br />
<br />
==[[Splitting Genes for HPP]]==<br />
<br />
== '''Resources / Citations'''==<br />
<br />
[https://2006.igem.org/wiki/index.php/The_What%27s_and_How%27s Davidson's Wet Lab Protocols]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/MWSU_protocols Missouri Western's Wet Lab Protocols]<br />
<br />
[http://gcat.davidson.edu/iGEM07/genesplitter.html Spliting Genes Web Tool]<br />
#'''DMW Part Numbers for 2007 are BBa_I715000 to BBa_I715999.''' <br />
#[http://partsregistry.org/Help:BioBrick_Part_Names How to Name a New Part]<br />
#[http://partsregistry.org/Add_a_Part_to_the_Registry Entering the Part to the Registry]<br />
#[http://partsregistry.org/Help:Part_Features How to Annotate a Part]<br />
<br />
Cool site for Breakfast<br />
[http://www.cut-the-knot.org/SimpleGames/Flipper.shtml]<br />
<br />
Karen Acker's paper describing GFP and TetA(c) with Hix insertions<br />
[http://www.bio.davidson.edu/Courses/Immunology/Students/spring2006/Acker/Acker_finalpaperGFP.doc]<br />
<br />
Bruce Henschen's paper describing one-time flippable Hix sites<br />
[http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bruce_Finalpaper.doc]<br />
<br />
Intro to Hamiltonian Path Problem and DNA<br />
[http://www.ams.org/featurecolumn/archive/dna-abc2.html]<br />
<br />
Adelman, LM. Molecular Computation of Solutions To Combinatorial Problems. Science. 11 November 1994. Vol. 266. no. 5187, pp. 1021 - 1024<br />
<br />
Ptashne, Mark. A Genetic Switch: Phage Lambda Revisited, Third Edition. New York. Cold Spring Harbor Laboratory Press: 2004.<br />
<br />
Sambrook and Russell. 2001. Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratry Press. Cold Spring Harbor, New York pg. 1.145. 2007 June.<br />
<br />
== '''Literature and Registry Research'''==<br />
<br />
'''Registry Search for Possible Promoters:'''<br />
<br />
BBa_J24669 --- arabinose induced<br />
<br />
BBa_R0082 --- Is upstream of the ompC porin gene<br />
<br />
BBa_R0074 --- Penl regulated<br />
<br />
BBa_I14017 --- P(Rhl)<br />
<br />
BBa_I14018 --- P (Bla) --> amp resistance<br />
<br />
BBa_J3902 --- Pr Fe (Pl + Pll rus operon)<br />
<br />
BBa_R0077 --- CinR --> thought to have own terminator<br />
<br />
BBa_R0078 --- CinR (no RBS)<br />
<br />
BBa_R0062 --- luxR & HSL regulated -- luxpR<br />
<br />
Possibly the use of Constitutive Promoter Family Members to strengthen other promoters.<br />
<br />
'''Literature Search for Polycistronic Genes on Plasmids'''<br />
<br />
Sol Operon<br />
http://www.jstage.jst.go.jp/article/bbb/71/1/58/_pdf<br />
<br />
Transfer (tra) Operon<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1347297&blobtype=pdf<br />
<br />
Oligopeptide Permease (opp)<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1087318&blobtype=pdf</div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-06-19T13:15:53Z<p>Macampbell: /* '''Resources / Citations''' */</p>
<hr />
<div>===Davidson & Missouri Western Team Logos, iGEM2006=== <br><br />
[[Image:EHOP.gif|thumb|300px|center| ]]<br />
<br><br />
[[Image:Ihop.PNG|thumb|300px|center| ]]<br />
<br><br />
<br />
----<br />
== '''Team Meeting Notes''' ==<br />
<br />
Western Meeting Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/WesternMeetingNotes]<br />
<br />
== '''Math Related Notes''' ==<br />
<br />
Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/MathMeetingNotes]<br />
<br />
== '''Students''' ==<br />
<br />
[[Image:Logo.gif]]<br />
<br />
• Will DeLoache, Junior Biology Major, [mailto:wideloache@davidson.edu]<br />
<br />
• Oyinade Adefuye, Senior Biology Major, [mailto:oyinadeadefuye@yahoo.com]<br />
<br />
• Jim Dickson, Junior Math and Economics Major, [mailto:jidickson@davidson.edu]<br />
<br />
• Amber Shoecraft, Math Major, [mailto:ashoecraft@jcsu.edu]<br />
<br />
• Andrew Martens, Senior Biology Major, [mailto:anmartens@davidson.edu]<br />
<br />
• Michael Waters, Sophomore Biology Major, [mailto:miwaters@davidson.edu]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
<br />
• Jordan Baumgardner, Junior Biology, Biochemistry/Molecular Biology Major, [mailto:jbaumgardner@missouriwestern.edu]<br />
<br />
• Ryan Chilcoat, Junior Biology Major (Health Sciences), [mailto:rchilcoat@missouriwestern.edu]<br />
<br />
• Tom Crowley, Senior Biochemisty/Molecular Biology Major, [mailto:stc8033@missouriwestern.edu]<br />
<br />
• Lane H. Heard, Central High School graduate, [mailto:axenmoon@hotmail.com]<br />
<br />
• Nickolaus Morton, Junior Chemistry Major, [mailto:nmorton@missouriwestern.edu]<br />
<br />
• Michelle Ritter, Junior Mathematics Major, [mailto:mrr5418@missouriwestern.edu]<br />
<br />
• Jessica Treece, Junior Biology Major (Health Sciences), [mailto:jtreece@missouriwestern.edu]<br />
<br />
• Matthew Unzicker, Senior Biochemistry/Molecular Biology Major, [mailto:mru8487@missouriwestern.edu]<br />
<br />
• Amanda Valencia, Senior Biochem/Molecular Biology Major, [mailto:avalencia@missouriwestern.edu]<br />
<br />
== '''Faculty''' ==<br />
[[Image:Logo.gif]]<br />
<br />
• Malcolm Campbell [http://www.bio.davidson.edu/people/macampbell/macampbell.html], Professor, Department of Biology, [mailto:macampbell@davidson.edu]<br />
<br />
• [http://www.bio.davidson.edu/people/kahaynes/kahaynes.html Karmella Haynes], Visiting Assistant Professor, Department of Biology, [mailto:kahaynes@davidson.edu]<br />
<br />
• Laurie Heyer [http://www.davidson.edu/math/heyer/], Associate Professor, Department of Mathematics, [mailto:laheyer@davidson.edu]<br />
<br />
Shipping Address: Malcolm Campbell, Biology Dept. Davidson College, 209 Ridge Road, Davidson, NC 28036 [(704) 894-2692]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
• Todd Eckdahl [http://staff.missouriwestern.edu/~eckdahl/], Professor, Department of Biology, [mailto:eckdahl@missouriwestern.edu]<br />
<br />
• Jeff Poet [http://staff.missouriwestern.edu/~poet/], Assistant Professor, Department of Computer Science, Mathematics, and Physics, [mailto:poet@missouriwestern.edu]<br />
<br />
Shipping Address: Todd Eckdahl, Biology Department, Missouri Western State University, 4525 Downs Drive, Saint Joseph, MO, 64507 [(816) 271-5873]<br />
<br />
== '''Project Overview'''==<br />
<br />
<font color="blue">Hamiltonian Path Problem</font color> <br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/hix DNA recombination mechanism which exists in nature in Salmonella as standard biobricks for use in E. coli. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we continue our collaboration and our efforts to manipulate E. coli into mathematics problem solvers as we refine our efforts with the hin/hix mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adelman in 1994 (citation below) where a unique Hamiltonian path was found in vitro for a particular directed graph on seven nodes. We propose to make progress toward solving the particular problem in vivo.<br />
<br />
[[Image:AdelmanGraph.JPG|thumb|300px|center| ]] <br />
<br><br />
<br />
<br />
<br />
==[[Splitting Genes for HPP]]==<br />
<br />
== '''Resources / Citations'''==<br />
<br />
[https://2006.igem.org/wiki/index.php/The_What%27s_and_How%27s Davidson's Wet Lab Protocols]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/MWSU_protocols Missouri Western's Wet Lab Protocols]<br />
<br />
[http://gcat.davidson.edu/iGEM07/genesplitter.html Spliting Genes Web Tool]<br />
<br />
Cool site for Breakfast<br />
[http://www.cut-the-knot.org/SimpleGames/Flipper.shtml]<br />
<br />
Karen Acker's paper describing GFP and TetA(c) with Hix insertions<br />
[http://www.bio.davidson.edu/Courses/Immunology/Students/spring2006/Acker/Acker_finalpaperGFP.doc]<br />
<br />
Bruce Henschen's paper describing one-time flippable Hix sites<br />
[http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bruce_Finalpaper.doc]<br />
<br />
Intro to Hamiltonian Path Problem and DNA<br />
[http://www.ams.org/featurecolumn/archive/dna-abc2.html]<br />
<br />
Adelman, LM. Molecular Computation of Solutions To Combinatorial Problems. Science. 11 November 1994. Vol. 266. no. 5187, pp. 1021 - 1024<br />
<br />
Ptashne, Mark. A Genetic Switch: Phage Lambda Revisited, Third Edition. New York. Cold Spring Harbor Laboratory Press: 2004.<br />
<br />
Sambrook and Russell. 2001. Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratry Press. Cold Spring Harbor, New York pg. 1.145. 2007 June.<br />
<br />
#'''DMW Part Numbers for 2007 are BBa_I715000 to BBa_I715999.''' <br />
#[http://partsregistry.org/Help:BioBrick_Part_Names How to Name a New Part]<br />
#[http://partsregistry.org/Add_a_Part_to_the_Registry Entering the Part to the Registry]<br />
#[http://partsregistry.org/Help:Part_Features How to Annotate a Part]<br />
<br />
== '''Literature and Registry Research'''==<br />
<br />
'''Registry Search for Possible Promoters:'''<br />
<br />
BBa_J24669 --- arabinose induced<br />
<br />
BBa_R0082 --- Is upstream of the ompC porin gene<br />
<br />
BBa_R0074 --- Penl regulated<br />
<br />
BBa_I14017 --- P(Rhl)<br />
<br />
BBa_I14018 --- P (Bla) --> amp resistance<br />
<br />
BBa_J3902 --- Pr Fe (Pl + Pll rus operon)<br />
<br />
BBa_R0077 --- CinR --> thought to have own terminator<br />
<br />
BBa_R0078 --- CinR (no RBS)<br />
<br />
BBa_R0062 --- luxR & HSL regulated -- luxpR<br />
<br />
Possibly the use of Constitutive Promoter Family Members to strengthen other promoters.<br />
<br />
'''Literature Search for Polycistronic Genes on Plasmids'''<br />
<br />
Sol Operon<br />
http://www.jstage.jst.go.jp/article/bbb/71/1/58/_pdf<br />
<br />
Transfer (tra) Operon<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1347297&blobtype=pdf<br />
<br />
Oligopeptide Permease (opp)<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1087318&blobtype=pdf</div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-06-19T12:59:20Z<p>Macampbell: /* '''Resources / Citations''' */</p>
<hr />
<div>===Davidson & Missouri Western Team Logos, iGEM2006=== <br><br />
[[Image:EHOP.gif|thumb|300px|center| ]]<br />
<br><br />
[[Image:Ihop.PNG|thumb|300px|center| ]]<br />
<br><br />
<br />
----<br />
== '''Team Meeting Notes''' ==<br />
<br />
Western Meeting Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/WesternMeetingNotes]<br />
<br />
== '''Math Related Notes''' ==<br />
<br />
Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/MathMeetingNotes]<br />
<br />
== '''Students''' ==<br />
<br />
[[Image:Logo.gif]]<br />
<br />
• Will DeLoache, Junior Biology Major, [mailto:wideloache@davidson.edu]<br />
<br />
• Oyinade Adefuye, Senior Biology Major, [mailto:oyinadeadefuye@yahoo.com]<br />
<br />
• Jim Dickson, Junior Math and Economics Major, [mailto:jidickson@davidson.edu]<br />
<br />
• Amber Shoecraft, Math Major, [mailto:ashoecraft@jcsu.edu]<br />
<br />
• Andrew Martens, Senior Biology Major, [mailto:anmartens@davidson.edu]<br />
<br />
• Michael Waters, Sophomore Biology Major, [mailto:miwaters@davidson.edu]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
<br />
• Jordan Baumgardner, Junior Biology, Biochemistry/Molecular Biology Major, [mailto:jbaumgardner@missouriwestern.edu]<br />
<br />
• Ryan Chilcoat, Junior Biology Major (Health Sciences), [mailto:rchilcoat@missouriwestern.edu]<br />
<br />
• Tom Crowley, Senior Biochemisty/Molecular Biology Major, [mailto:stc8033@missouriwestern.edu]<br />
<br />
• Lane H. Heard, Central High School graduate, [mailto:axenmoon@hotmail.com]<br />
<br />
• Nickolaus Morton, Junior Chemistry Major, [mailto:nmorton@missouriwestern.edu]<br />
<br />
• Michelle Ritter, Junior Mathematics Major, [mailto:mrr5418@missouriwestern.edu]<br />
<br />
• Jessica Treece, Junior Biology Major (Health Sciences), [mailto:jtreece@missouriwestern.edu]<br />
<br />
• Matthew Unzicker, Senior Biochemistry/Molecular Biology Major, [mailto:mru8487@missouriwestern.edu]<br />
<br />
• Amanda Valencia, Senior Biochem/Molecular Biology Major, [mailto:avalencia@missouriwestern.edu]<br />
<br />
== '''Faculty''' ==<br />
[[Image:Logo.gif]]<br />
<br />
• Malcolm Campbell [http://www.bio.davidson.edu/people/macampbell/macampbell.html], Professor, Department of Biology, [mailto:macampbell@davidson.edu]<br />
<br />
• [http://www.bio.davidson.edu/people/kahaynes/kahaynes.html Karmella Haynes], Visiting Assistant Professor, Department of Biology, [mailto:kahaynes@davidson.edu]<br />
<br />
• Laurie Heyer [http://www.davidson.edu/math/heyer/], Associate Professor, Department of Mathematics, [mailto:laheyer@davidson.edu]<br />
<br />
Shipping Address: Malcolm Campbell, Biology Dept. Davidson College, 209 Ridge Road, Davidson, NC 28036 [(704) 894-2692]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
• Todd Eckdahl [http://staff.missouriwestern.edu/~eckdahl/], Professor, Department of Biology, [mailto:eckdahl@missouriwestern.edu]<br />
<br />
• Jeff Poet [http://staff.missouriwestern.edu/~poet/], Assistant Professor, Department of Computer Science, Mathematics, and Physics, [mailto:poet@missouriwestern.edu]<br />
<br />
Shipping Address: Todd Eckdahl, Biology Department, Missouri Western State University, 4525 Downs Drive, Saint Joseph, MO, 64507 [(816) 271-5873]<br />
<br />
== '''Project Overview'''==<br />
<br />
<font color="blue">Hamiltonian Path Problem</font color> <br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/hix DNA recombination mechanism which exists in nature in Salmonella as standard biobricks for use in E. coli. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we continue our collaboration and our efforts to manipulate E. coli into mathematics problem solvers as we refine our efforts with the hin/hix mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adelman in 1994 (citation below) where a unique Hamiltonian path was found in vitro for a particular directed graph on seven nodes. We propose to make progress toward solving the particular problem in vivo.<br />
<br />
[[Image:AdelmanGraph.JPG|thumb|300px|center| ]] <br />
<br><br />
<br />
<br />
<br />
==[[Splitting Genes for HPP]]==<br />
<br />
== '''Resources / Citations'''==<br />
<br />
[https://2006.igem.org/wiki/index.php/The_What%27s_and_How%27s Davidson's Wet Lab Protocols]<br />
<br />
[https://2007.igem.org/Davidson_Missouri_W/MWSU_protocols Missouri Western's Wet Lab Protocols]<br />
<br />
[http://gcat.davidson.edu/iGEM07/genesplitter.html Spliting Genes Web Tool]<br />
<br />
Cool site for Breakfast<br />
[http://www.cut-the-knot.org/SimpleGames/Flipper.shtml]<br />
<br />
Karen Acker's paper describing GFP and TetA(c) with Hix insertions<br />
[http://www.bio.davidson.edu/Courses/Immunology/Students/spring2006/Acker/Acker_finalpaperGFP.doc]<br />
<br />
Bruce Henschen's paper describing one-time flippable Hix sites<br />
[http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bruce_Finalpaper.doc]<br />
<br />
Intro to Hamiltonian Path Problem and DNA<br />
[http://www.ams.org/featurecolumn/archive/dna-abc2.html]<br />
<br />
Adelman, LM. Molecular Computation of Solutions To Combinatorial Problems. Science. 11 November 1994. Vol. 266. no. 5187, pp. 1021 - 1024<br />
<br />
Ptashne, Mark. A Genetic Switch: Phage Lambda Revisited, Third Edition. New York. Cold Spring Harbor Laboratory Press: 2004.<br />
<br />
Sambrook and Russell. 2001. Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratry Press. Cold Spring Harbor, New York pg. 1.145. 2007 June.<br />
<br />
#[http://partsregistry.org/Help:BioBrick_Part_Names How to Name a New Part]<br />
#[http://partsregistry.org/Add_a_Part_to_the_Registry Entering the Part to the Registry]<br />
#[http://partsregistry.org/Help:Part_Features How to Annotate a Part]<br />
<br />
== '''Literature and Registry Research'''==<br />
<br />
'''Registry Search for Possible Promoters:'''<br />
<br />
BBa_J24669 --- arabinose induced<br />
<br />
BBa_R0082 --- Is upstream of the ompC porin gene<br />
<br />
BBa_R0074 --- Penl regulated<br />
<br />
BBa_I14017 --- P(Rhl)<br />
<br />
BBa_I14018 --- P (Bla) --> amp resistance<br />
<br />
BBa_J3902 --- Pr Fe (Pl + Pll rus operon)<br />
<br />
BBa_R0077 --- CinR --> thought to have own terminator<br />
<br />
BBa_R0078 --- CinR (no RBS)<br />
<br />
BBa_R0062 --- luxR & HSL regulated -- luxpR<br />
<br />
Possibly the use of Constitutive Promoter Family Members to strengthen other promoters.<br />
<br />
'''Literature Search for Polycistronic Genes on Plasmids'''<br />
<br />
Sol Operon<br />
http://www.jstage.jst.go.jp/article/bbb/71/1/58/_pdf<br />
<br />
Transfer (tra) Operon<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1347297&blobtype=pdf<br />
<br />
Oligopeptide Permease (opp)<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1087318&blobtype=pdf</div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_WDavidson Missouri W2007-06-19T12:55:08Z<p>Macampbell: /* '''Resources / Citations''' */</p>
<hr />
<div>===Davidson & Missouri Western Team Logos, iGEM2006=== <br><br />
[[Image:EHOP.gif|thumb|300px|center| ]]<br />
<br><br />
[[Image:Ihop.PNG|thumb|300px|center| ]]<br />
<br><br />
<br />
----<br />
== '''Team Meeting Notes''' ==<br />
<br />
Western Meeting Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/WesternMeetingNotes]<br />
<br />
== '''Math Related Notes''' ==<br />
<br />
Notes 051407 to Present<br />
[https://2007.igem.org/Davidson_Missouri_W/MathMeetingNotes]<br />
<br />
== '''Students''' ==<br />
<br />
[[Image:Logo.gif]]<br />
<br />
• Will DeLoache, Junior Biology Major, [mailto:wideloache@davidson.edu]<br />
<br />
• Oyinade Adefuye, Senior Biology Major, [mailto:oyinadeadefuye@yahoo.com]<br />
<br />
• Jim Dickson, Junior Math and Economics Major, [mailto:jidickson@davidson.edu]<br />
<br />
• Amber Shoecraft, Math Major, [mailto:ashoecraft@jcsu.edu]<br />
<br />
• Andrew Martens, Senior Biology Major, [mailto:anmartens@davidson.edu]<br />
<br />
• Michael Waters, Sophomore Biology Major, [mailto:miwaters@davidson.edu]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
<br />
• Jordan Baumgardner, Junior Biology, Biochemistry/Molecular Biology Major, [mailto:jbaumgardner@missouriwestern.edu]<br />
<br />
• Ryan Chilcoat, Junior Biology Major (Health Sciences), [mailto:rchilcoat@missouriwestern.edu]<br />
<br />
• Tom Crowley, Senior Biochemisty/Molecular Biology Major, [mailto:stc8033@missouriwestern.edu]<br />
<br />
• Lane H. Heard, Central High School graduate, [mailto:axenmoon@hotmail.com]<br />
<br />
• Nickolaus Morton, Junior Chemistry Major, [mailto:nmorton@missouriwestern.edu]<br />
<br />
• Michelle Ritter, Junior Mathematics Major, [mailto:mrr5418@missouriwestern.edu]<br />
<br />
• Jessica Treece, Junior Biology Major (Health Sciences), [mailto:jtreece@missouriwestern.edu]<br />
<br />
• Matthew Unzicker, Senior Biochemistry/Molecular Biology Major, [mailto:mru8487@missouriwestern.edu]<br />
<br />
• Amanda Valencia, Senior Biochem/Molecular Biology Major, [mailto:avalencia@missouriwestern.edu]<br />
<br />
== '''Faculty''' ==<br />
[[Image:Logo.gif]]<br />
<br />
• Malcolm Campbell [http://www.bio.davidson.edu/people/macampbell/macampbell.html], Professor, Department of Biology, [mailto:macampbell@davidson.edu]<br />
<br />
• [http://www.bio.davidson.edu/people/kahaynes/kahaynes.html Karmella Haynes], Visiting Assistant Professor, Department of Biology, [mailto:kahaynes@davidson.edu]<br />
<br />
• Laurie Heyer [http://www.davidson.edu/math/heyer/], Associate Professor, Department of Mathematics, [mailto:laheyer@davidson.edu]<br />
<br />
Shipping Address: Malcolm Campbell, Biology Dept. Davidson College, 209 Ridge Road, Davidson, NC 28036 [(704) 894-2692]<br />
<br />
-----<br />
<br />
[[Image:Header_01.gif]]<br />
<br />
• Todd Eckdahl [http://staff.missouriwestern.edu/~eckdahl/], Professor, Department of Biology, [mailto:eckdahl@missouriwestern.edu]<br />
<br />
• Jeff Poet [http://staff.missouriwestern.edu/~poet/], Assistant Professor, Department of Computer Science, Mathematics, and Physics, [mailto:poet@missouriwestern.edu]<br />
<br />
Shipping Address: Todd Eckdahl, Biology Department, Missouri Western State University, 4525 Downs Drive, Saint Joseph, MO, 64507 [(816) 271-5873]<br />
<br />
== '''Project Overview'''==<br />
<br />
<font color="blue">Hamiltonian Path Problem</font color> <br />
As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/hix DNA recombination mechanism which exists in nature in Salmonella as standard biobricks for use in E. coli. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions.<br />
<br />
For 2007, we continue our collaboration and our efforts to manipulate E. coli into mathematics problem solvers as we refine our efforts with the hin/hix mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adelman in 1994 (citation below) where a unique Hamiltonian path was found in vitro for a particular directed graph on seven nodes. We propose to make progress toward solving the particular problem in vivo.<br />
<br />
[[Image:AdelmanGraph.JPG|thumb|300px|center| ]] <br />
<br><br />
<br />
<br />
<br />
==[[Splitting Genes for HPP]]==<br />
<br />
== '''Resources / Citations'''==<br />
<br />
*[https://2006.igem.org/wiki/index.php/The_What%27s_and_How%27s Davidson's Wet Lab Protocols]<br />
<br />
*[https://2007.igem.org/Davidson_Missouri_W/MWSU_protocols Missouri Western's Wet Lab Protocols]<br />
<br />
*[http://gcat.davidson.edu/iGEM07/genesplitter.html Spliting Genes Web Tool]<br />
<br />
*[http://partsregistry.org/Help:Plasmids Plasmid Naming System]<br />
<br />
Cool site for Breakfast<br />
[http://www.cut-the-knot.org/SimpleGames/Flipper.shtml]<br />
<br />
Karen Acker's paper describing GFP and TetA(c) with Hix insertions<br />
[http://www.bio.davidson.edu/Courses/Immunology/Students/spring2006/Acker/Acker_finalpaperGFP.doc]<br />
<br />
Bruce Henschen's paper describing one-time flippable Hix sites<br />
[http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bruce_Finalpaper.doc]<br />
<br />
Intro to Hamiltonian Path Problem and DNA<br />
[http://www.ams.org/featurecolumn/archive/dna-abc2.html]<br />
<br />
Adelman, LM. Molecular Computation of Solutions To Combinatorial Problems. Science. 11 November 1994. Vol. 266. no. 5187, pp. 1021 - 1024<br />
<br />
Ptashne, Mark. A Genetic Switch: Phage Lambda Revisited, Third Edition. New York. Cold Spring Harbor Laboratory Press: 2004.<br />
<br />
Sambrook and Russell. 2001. Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratry Press. Cold Spring Harbor, New York pg. 1.145. 2007 June.<br />
<br />
== '''Literature and Registry Research'''==<br />
<br />
'''Registry Search for Possible Promoters:'''<br />
<br />
BBa_J24669 --- arabinose induced<br />
<br />
BBa_R0082 --- Is upstream of the ompC porin gene<br />
<br />
BBa_R0074 --- Penl regulated<br />
<br />
BBa_I14017 --- P(Rhl)<br />
<br />
BBa_I14018 --- P (Bla) --> amp resistance<br />
<br />
BBa_J3902 --- Pr Fe (Pl + Pll rus operon)<br />
<br />
BBa_R0077 --- CinR --> thought to have own terminator<br />
<br />
BBa_R0078 --- CinR (no RBS)<br />
<br />
BBa_R0062 --- luxR & HSL regulated -- luxpR<br />
<br />
Possibly the use of Constitutive Promoter Family Members to strengthen other promoters.<br />
<br />
'''Literature Search for Polycistronic Genes on Plasmids'''<br />
<br />
Sol Operon<br />
http://www.jstage.jst.go.jp/article/bbb/71/1/58/_pdf<br />
<br />
Transfer (tra) Operon<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1347297&blobtype=pdf<br />
<br />
Oligopeptide Permease (opp)<br />
http://www.pubmedcentral.nig.gov/picrender.fcgi?artid=1087318&blobtype=pdf</div>Macampbellhttp://2007.igem.org/wiki/index.php/Splitting_Genes_for_HPPSplitting Genes for HPP2007-06-18T21:37:39Z<p>Macampbell: </p>
<hr />
<div>We have selected 4 genes to split. We will use our [http://gcat.davidson.edu/iGEM07/genesplitter.html online gene splitting tool] to choose the PCR primers. Davidson will produce these 4 split genes and test each one. <br />
<br />
# Red Fluorescent Protein<br />
# Kanamycin Nucleotidyltransferase <br />
# Cre Recombinase<br />
# Chloramphenicol Acetyltransferase<br />
<br />
<br />
We will also produce two constructs for tetsing promoters. MWSU will produec (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes. <br />
<br />
Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and lack the backwards promotion we have detected with Part: [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. We will test out the modified promoter [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] which is reported to have good repression and strong induction. We may still introduce the UV5 double mutation to enhance transcription and compare with R0010.<br />
<br />
MWSU is also going to produce backwards LacIq to put upstream of pLac [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. The purpose of this is to have more LacIq in the cytoplasm at all times, regardless of ITPG status. <br />
<br />
MWSU is also testing two of the [http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bio372.html one-time-flippable HixC sites] produced by Bruce spring 2007. <br />
<br />
MWSU is going to produce a new Hin-LVA expression plasmid that has ColE1 ori and uses Tet as the selectable marker. <br />
<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br><br />
----<br />
'''DsRed - Red Fluorescent Protein'''<br />
<br />
We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 ''Discosoma sp.'']), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.<br />
<br />
Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.<br />
<center><br />
[[Image:Rfp hix insertion point.jpg]]<br />
</center><br />
<br><br />
----<br />
<br />
'''Kanamycin Nucleotidyltransferase'''<br />
<br />
One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA.[http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1] <br />
Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimmer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that: <br />
<br />
-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]<br />
<br />
-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-We did not consider any Amino Acids near the N or C terminus <br />
<br />
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)<br />
<br />
<center><br />
[[Image:KNTase hix cut.png]]<br />
<br />
The yellow bands at the top and bottom of the molecule denotes hix site insertion<br />
</center><br />
<br />
We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.<br />
<br><br />
----<br />
<br />
'''Chloramphenicol Acetyltransferase'''<br />
<br />
<br />
[http://partsregistry.org/Part:BBa_P1004 Chloramphenicol Acetyltransferase] was one of the genes we chose as a node for our Hamiltonian Path. It is a bacterial gene that neutralizes the effect of an antibiotic, Chloramphenicol, by transferring acetyl groups to Chloramphenicol and changes its shape into a harmless form. <br />
<br />
The specific Chloramphenicol Acetyltransferase gene we are using comes from the plasmid PSV2CAT whose original source is an ''E. coli'' transposable element Tn9 (Sambrook, 2001) Its PDB ID# is [http://www.pdb.org/pdb/explore/explore.do?structureId=1PD5 1PD5]. <br />
<br />
I have chosen to insert my hixC site between amino acid 52 and 53. I chose this point because it is away from the active site of the protein, the point that contains the catalytic binding sites and allow the recognition and binding of the substrate. It is important for the insertion point to be away from the active site because we do not want the overall function and structure of the protein to be destroyed in the process of splitting. We want to split at a point where the two halves of the protein cannot work as single units, but once a hixC site has been inserted, and the two halves are brought back together, the protein displays its original function.<br />
<center><br />
[[Image: 1pd5.jpg]]<br />
<br />
<br />
The structure of a type I Chloramphenicol Acetyltransferase used in the BioBrick Registry. <br />
<br />
<br />
[[Image: type 3.jpg]]<br />
<br />
The structure of a type III Chloramphenicol Acetyltransferase. <br />
</center><br />
<br />
This structure shows the active sites of the protein. Although the two proteins are of different types and the amino acid sequence are different, the structures are similar and we can therefore see that the point where we have decided to split is “safe”. The exact points that I have decided to cut is away from the active site. <br />
<br />
<br />
<br />
<br><br />
----<br />
<br />
'''Cre Recombinase'''<br />
<br />
[[Image:Cre recombinase monomer.png]]<br />
<br />
[[Image:Cre recombinase tetramer.png]]<br />
<br />
[http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT?CRETRY=1&SRETRY=0].<br />
<br />
<br><br />
----<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br></div>Macampbellhttp://2007.igem.org/wiki/index.php/Splitting_Genes_for_HPPSplitting Genes for HPP2007-06-18T21:36:55Z<p>Macampbell: </p>
<hr />
<div>We have selected 4 genes to split. We will use our [http://gcat.davidson.edu/iGEM07/genesplitter.html online gene splitting tool] to choose the PCR primers. Davidson will produce these 4 split genes and test each one. <br />
<br />
# Red Fluorescent Protein<br />
# Kanamycin Nucleotidyltransferase <br />
# Cre Recombinase<br />
# Chloramphenicol Acetyltransferase<br />
<br />
<br />
We will also produce two constructs for tetsing promoters. MWSU will produec (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes. <br />
<br />
Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and lack the backwards promotion we have detected with Part: [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. We will test out the modified promoter [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] which is reported to have good repression and strong induction. We may still introduce the UV5 double mutation to enhance transcription and compare with R0010.<br />
<br />
MWSU is also going to produce backwards LacIq to put upstream of pLac [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. The purpose of this is to have more LacIq in the cytoplasm at all times, regardless of ITPG status. <br />
<br />
MWSU is also testing two of the [http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bio372.html one-time-flippable HixC sites] produced by Bruce spring 2007. <br />
<br />
MWSU is going to produce a new Hin-LVA expression plasmid that has ColE1 ori and uses Tet as the selectable marker. <br />
<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br><br />
----<br />
'''DsRed - Red Fluorescent Protein'''<br />
<br />
We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 ''Discosoma sp.'']), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.<br />
<br />
Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.<br />
<center><br />
[[Image:Rfp hix insertion point.jpg]]<br />
</center><br />
<br><br />
----<br />
<br />
'''Kanamycin Nucleotidyltransferase'''<br />
<br />
One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA.[http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1] <br />
Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimmer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that: <br />
<br />
-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]<br />
<br />
-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-We did not consider any Amino Acids near the N or C terminus <br />
<br />
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)<br />
<center><br />
[[Image:KNTase hix cut.png]]<br />
</center><br />
The yellow bands at the top and bottom of the molecule denotes hix site insertion<br />
<br />
We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.<br />
<br><br />
----<br />
<br />
'''Chloramphenicol Acetyltransferase'''<br />
<br />
<br />
[http://partsregistry.org/Part:BBa_P1004 Chloramphenicol Acetyltransferase] was one of the genes we chose as a node for our Hamiltonian Path. It is a bacterial gene that neutralizes the effect of an antibiotic, Chloramphenicol, by transferring acetyl groups to Chloramphenicol and changes its shape into a harmless form. <br />
<br />
The specific Chloramphenicol Acetyltransferase gene we are using comes from the plasmid PSV2CAT whose original source is an ''E. coli'' transposable element Tn9 (Sambrook, 2001) Its PDB ID# is [http://www.pdb.org/pdb/explore/explore.do?structureId=1PD5 1PD5]. <br />
<br />
I have chosen to insert my hixC site between amino acid 52 and 53. I chose this point because it is away from the active site of the protein, the point that contains the catalytic binding sites and allow the recognition and binding of the substrate. It is important for the insertion point to be away from the active site because we do not want the overall function and structure of the protein to be destroyed in the process of splitting. We want to split at a point where the two halves of the protein cannot work as single units, but once a hixC site has been inserted, and the two halves are brought back together, the protein displays its original function.<br />
<center><br />
[[Image: 1pd5.jpg]]<br />
<br />
<br />
The structure of a type I Chloramphenicol Acetyltransferase used in the BioBrick Registry. <br />
<br />
<br />
[[Image: type 3.jpg]]<br />
<br />
The structure of a type III Chloramphenicol Acetyltransferase. <br />
</center><br />
<br />
This structure shows the active sites of the protein. Although the two proteins are of different types and the amino acid sequence are different, the structures are similar and we can therefore see that the point where we have decided to split is “safe”. The exact points that I have decided to cut is away from the active site. <br />
<br />
<br />
<br />
<br><br />
----<br />
<br />
'''Cre Recombinase'''<br />
<br />
[[Image:Cre recombinase monomer.png]]<br />
<br />
[[Image:Cre recombinase tetramer.png]]<br />
<br />
[http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT?CRETRY=1&SRETRY=0].<br />
<br />
<br><br />
----<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br></div>Macampbellhttp://2007.igem.org/wiki/index.php/Splitting_Genes_for_HPPSplitting Genes for HPP2007-06-18T21:36:02Z<p>Macampbell: </p>
<hr />
<div>We have selected 4 genes to split. We will use our [http://gcat.davidson.edu/iGEM07/genesplitter.html online gene splitting tool] to choose the PCR primers. Davidson will produce these 4 split genes and test each one. <br />
<br />
# Red Fluorescent Protein<br />
# Kanamycin Nucleotidyltransferase <br />
# Cre Recombinase<br />
# Chloramphenicol Acetyltransferase<br />
<br />
<br />
We will also produce two constructs for tetsing promoters. MWSU will produec (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes. <br />
<br />
Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and lack the backwards promotion we have detected with Part: [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. We will test out the modified promoter [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] which is reported to have good repression and strong induction. We may still introduce the UV5 double mutation to enhance transcription and compare with R0010.<br />
<br />
MWSU is also going to produce backwards LacIq to put upstream of pLac [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. The purpose of this is to have more LacIq in the cytoplasm at all times, regardless of ITPG status. <br />
<br />
MWSU is also testing two of the [http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bio372.html one-time-flippable HixC sites] produced by Bruce spring 2007. <br />
<br />
MWSU is going to produce a new Hin-LVA expression plasmid that has ColE1 ori and uses Tet as the selectable marker. <br />
<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br><br />
----<br />
'''DsRed - Red Fluorescent Protein'''<br />
<br />
We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 ''Discosoma sp.'']), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.<br />
<br />
Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.<br />
<br />
[[Image:Rfp hix insertion point.jpg]]<br />
<br><br />
----<br />
<br />
'''Kanamycin Nucleotidyltransferase'''<br />
<br />
One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA.[http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1] <br />
Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimmer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that: <br />
<br />
-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]<br />
<br />
-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-We did not consider any Amino Acids near the N or C terminus <br />
<br />
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)<br />
<br />
[[Image:KNTase hix cut.png]]<br />
<br />
The yellow bands at the top and bottom of the molecule denotes hix site insertion<br />
<br />
We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.<br />
<br><br />
----<br />
<br />
'''Chloramphenicol Acetyltransferase'''<br />
<br />
<br />
[http://partsregistry.org/Part:BBa_P1004 Chloramphenicol Acetyltransferase] was one of the genes we chose as a node for our Hamiltonian Path. It is a bacterial gene that neutralizes the effect of an antibiotic, Chloramphenicol, by transferring acetyl groups to Chloramphenicol and changes its shape into a harmless form. <br />
<br />
The specific Chloramphenicol Acetyltransferase gene we are using comes from the plasmid PSV2CAT whose original source is an ''E. coli'' transposable element Tn9 (Sambrook, 2001) Its PDB ID# is [http://www.pdb.org/pdb/explore/explore.do?structureId=1PD5 1PD5]. <br />
<br />
I have chosen to insert my hixC site between amino acid 52 and 53. I chose this point because it is away from the active site of the protein, the point that contains the catalytic binding sites and allow the recognition and binding of the substrate. It is important for the insertion point to be away from the active site because we do not want the overall function and structure of the protein to be destroyed in the process of splitting. We want to split at a point where the two halves of the protein cannot work as single units, but once a hixC site has been inserted, and the two halves are brought back together, the protein displays its original function.<br />
<center><br />
[[Image: 1pd5.jpg]]<br />
<br />
<br />
The structure of a type I Chloramphenicol Acetyltransferase used in the BioBrick Registry. <br />
<br />
<br />
[[Image: type 3.jpg]]<br />
<br />
The structure of a type III Chloramphenicol Acetyltransferase. <br />
</center><br />
<br />
This structure shows the active sites of the protein. Although the two proteins are of different types and the amino acid sequence are different, the structures are similar and we can therefore see that the point where we have decided to split is “safe”. The exact points that I have decided to cut is away from the active site. <br />
<br />
<br />
<br />
<br><br />
----<br />
<br />
'''Cre Recombinase'''<br />
<br />
[[Image:Cre recombinase monomer.png]]<br />
<br />
[[Image:Cre recombinase tetramer.png]]<br />
<br />
[http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT?CRETRY=1&SRETRY=0].<br />
<br />
<br><br />
----<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br></div>Macampbellhttp://2007.igem.org/wiki/index.php/Splitting_Genes_for_HPPSplitting Genes for HPP2007-06-18T21:34:16Z<p>Macampbell: </p>
<hr />
<div>We have selected 4 genes to split. We will use our [http://gcat.davidson.edu/iGEM07/genesplitter.html online gene splitting tool] to choose the PCR primers. Davidson will produce these 4 split genes and test each one. <br />
<br />
# Red Fluorescent Protein<br />
# Kanamycin Nucleotidyltransferase <br />
# Cre Recombinase<br />
# Chloramphenicol Acetyltransferase<br />
<br />
<br />
We will also produce two constructs for tetsing promoters. MWSU will produec (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes. <br />
<br />
Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and lack the backwards promotion we have detected with Part: [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. We will test out the modified promoter [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] which is reported to have good repression and strong induction. We may still introduce the UV5 double mutation to enhance transcription and compare with R0010.<br />
<br />
MWSU is also going to produce backwards LacIq to put upstream of pLac [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. The purpose of this is to have more LacIq in the cytoplasm at all times, regardless of ITPG status. <br />
<br />
MWSU is also testing two of the [http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bio372.html one-time-flippable HixC sites] produced by Bruce spring 2007. <br />
<br />
MWSU is going to produce a new Hin-LVA expression plasmid that has ColE1 ori and uses Tet as the selectable marker. <br />
<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br><br />
----<br />
'''DsRed - Red Fluorescent Protein'''<br />
<br />
We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 ''Discosoma sp.'']), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.<br />
<br />
Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.<br />
<br />
[[Image:Rfp hix insertion point.jpg]]<br />
<br><br />
----<br />
<br />
'''Kanamycin Nucleotidyltransferase'''<br />
<br />
One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA.[http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1] <br />
Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimmer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that: <br />
<br />
-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]<br />
<br />
-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-We did not consider any Amino Acids near the N or C terminus <br />
<br />
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)<br />
<br />
[[Image:KNTase hix cut.png]]<br />
<br />
The yellow bands at the top and bottom of the molecule denotes hix site insertion<br />
<br />
We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.<br />
<br><br />
----<br />
<br />
'''Chloramphenicol Acetyltransferase'''<br />
<br />
<br />
[http://partsregistry.org/Part:BBa_P1004 Chloramphenicol Acetyltransferase] was one of the genes we chose as a node for our Hamiltonian Path. It is a bacterial gene that neutralizes the effect of an antibiotic, Chloramphenicol, by transferring acetyl groups to Chloramphenicol and changes its shape into a harmless form. <br />
<br />
The specific Chloramphenicol Acetyltransferase gene we are using comes from the plasmid PSV2CAT whose original source is an E coli transposable element Tn9 (Sambrook, 2001) Its PDB ID# is [http://www.pdb.org/pdb/explore/explore.do?structureId=1PD5 1PD5]. <br />
<br />
I have chosen to insert my hixC site between amino acid 52 and 53. I chose this point because it is away from the active site of the protein, the point that contains the catalytic binding sites and allow the recognition and binding of the substrate. It is important for the insertion point to be away from the active site because we do not want the overall function and structure of the protein to be destroyed in the process of splitting. We want to split at a point where the two halves of the protein cannot work as single units, but once a hixC site has been inserted, and the two halves are brought back together, the protein displays its original function.<br />
<center><br />
[[Image: 1pd5.jpg]]<br />
</center><br />
<br />
The structure of a type I Chloramphenicol Acetyltransferase that we would be making use of<br />
<br />
<br />
<center><br />
[[Image: type 3.jpg]]<br />
</center><br />
<br />
The structure of a type III Chloramphenicol Acetyltransferase. This structure shows the active sites of the protein. Although the two proteins are of different types and the amino acid sequence are different, the structures are similar and we can therefore see that the point where we have decided to split is “safe”. The exact points that I have decided to cut is away from the active site. <br />
<br />
<br />
<br />
<br><br />
----<br />
<br />
'''Cre Recombinase'''<br />
<br />
[[Image:Cre recombinase monomer.png]]<br />
<br />
[[Image:Cre recombinase tetramer.png]]<br />
<br />
[http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT?CRETRY=1&SRETRY=0].<br />
<br />
<br><br />
----<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br></div>Macampbellhttp://2007.igem.org/wiki/index.php/Splitting_Genes_for_HPPSplitting Genes for HPP2007-06-18T21:28:56Z<p>Macampbell: </p>
<hr />
<div>We have selected 4 genes to split. We will use our [http://gcat.davidson.edu/iGEM07/genesplitter.html online gene splitting tool] to choose the PCR primers. Davidson will produce these 4 split genes and test each one. <br />
<br />
# Red Fluorescent Protein<br />
# Kanamycin Nucleotidyltransferase <br />
# Cre Recombinase<br />
# Chloramphenicol Acetyltransferase<br />
<br />
<br />
We will also produce two constructs for tetsing promoters. MWSU will produec (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes. <br />
<br />
Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and lack the backwards promotion we have detected with Part: [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. We will test out the modified promoter [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] which is reported to have good repression and strong induction. We may still introduce the UV5 double mutation to enhance transcription and compare with R0010.<br />
<br />
MWSU is also going to produce backwards LacIq to put upstream of pLac [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. The purpose of this is to have more LacIq in the cytoplasm at all times, regardless of ITPG status. <br />
<br />
MWSU is also testing two of the [http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bio372.html one-time-flippable HixC sites] produced by Bruce spring 2007. <br />
<br />
MWSU is going to produce a new Hin-LVA expression plasmid that has ColE1 ori and uses Tet as the selectable marker. <br />
<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br><br />
----<br />
'''DsRed - Red Fluorescent Protein'''<br />
<br />
We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 ''Discosoma sp.'']), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.<br />
<br />
Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.<br />
<br />
[[Image:Rfp hix insertion point.jpg]]<br />
<br><br />
----<br />
<br />
'''Kanamycin Nucleotidyltransferase'''<br />
<br />
One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA.[http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1] <br />
Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimmer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that: <br />
<br />
-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]<br />
<br />
-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-We did not consider any Amino Acids near the N or C terminus <br />
<br />
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)<br />
<br />
[[Image:KNTase hix cut.png]]<br />
<br />
The yellow bands at the top and bottom of the molecule denotes hix site insertion<br />
<br />
We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.<br />
<br><br />
----<br />
<br />
'''Chloramphenicol Acetyltransferase'''<br />
<br />
<br />
Chloramphenicol Acetyltransferase was one of the genes we chose as a node for our Hamiltonian Path. It is a bacterial gene that neutralizes the effect of an antibiotic, Chloramphenicol, by transferring acetyl groups to Chloramphenicol and changes its shape into a harmless form. <br />
<br />
The specific Chloramphenicol Acetyltransferase gene we are using comes from the plasmid PSV2CAT whose original source is an E coli transposable element Tn9 (Sambrook, 2001) Its PDB # is 1PD5. <br />
<br />
I have chosen to insert my hixC site between amino acid 52 and 53. I chose this point because it is away from the active site of the protein, the point that contains the catalytic binding sites and allow the recognition and binding of the substrate. It is important for the insertion point to be away from the active site because we do not want the overall function and structure of the protein to be destroyed in the process of splitting. We want to split at a point where the two halves of the protein cannot work as single units, but once a hixC site has been inserted, and the two halves are brought back together, the protein displays its original function.<br />
<br />
[[Image: 1pd5.jpg]]<br />
<br />
<br />
The structure of a type I Chloramphenicol Acetyltransferase that we would be making use of<br />
<br />
<br />
<br />
[[Image: type 3.jpg]]<br />
<br />
<br />
The structure of a type III Chloramphenicol Acetyltransferase. This structure shows the active sites of the protein. Although the two proteins are of different types and the amino acid sequence are different, the structures are similar and we can therefore see that the point where we have decided to split is “safe”.<br />
<br />
<br />
<br />
[[Image: big 1pd5.jpg]]<br />
<br />
This image shows the exact points that I have decided to cut (in yellow). We can see that these points are away from the active sites and are not going to destroy any part of the protein during the process of hixC insertion.<br />
<br />
<br />
<br />
Nucleotide Sequence of Chloramphenicol Acetyltransferase obtained from the Bio Brick<br />
<br />
<br />
atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctata<br />
accagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgat<br />
gaatgctcatccggaatttcgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaa<br />
acgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatt<br />
tccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttctt<br />
cgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgatggcttccat<br />
gtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaa<br />
<br />
<br />
protein sequence gotten from our gene splitting tool:<br />
<br />
MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLD ITAFLKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDG ELVIWDSVHPCYTVFHEQTETFSSLWSEYHDDFRQFLHIY SQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNV ANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFH <br />
VGRMLNELQQYCDEWQGGA<br />
<br />
<br />
Here is AA sequence for PDB ID# 1PD5<br />
<br />
MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLD<br />
ITAFLKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDG<br />
ELVIWDSVHPCYTVFHEQTETFSSLWSEYHDDFRQFLHIY<br />
SQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNV<br />
ANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFH<br />
VGRMLNELQQYCDEWQGGA<br />
<br />
The above Amino Acid sequences confirm that the two proteins are 100% identical.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br />
'''Cre Recombinase'''<br />
<br />
[[Image:Cre recombinase monomer.png]]<br />
<br />
[[Image:Cre recombinase tetramer.png]]<br />
<br />
[http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT?CRETRY=1&SRETRY=0].<br />
<br />
<br><br />
----<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br></div>Macampbellhttp://2007.igem.org/wiki/index.php/Davidson_Missouri_W/colony_PCRDavidson Missouri W/colony PCR2007-06-18T17:59:25Z<p>Macampbell: </p>
<hr />
<div>This procedure uses PCR to determine the size of DNA cloned into a plasmid from a single colony on a transformation plate, while reserving some of the bacteria for further growth and plasmid preparation.<br />
<br />
1. Determine the number of colonies to be tested. Plan to conduct PCR on control plasmids with and without the insert. Assemble the following PCR mixture:<br />
<br />
Per Reaction<br />
<br />
*2 ul 10X reaction buffer<br />
*2 ul 2 mM dNTPs (working concentration 200 uM each)<br />
*1 ul forward primer G0100 (20 pmol)<br />
*1 ul reverse primer G0100 (20 pmol)<br />
*1 ul Taq DNA polymerase (2.5 u)<br />
*12 ul dH2O<br />
<br />
*19 ul total<br />
<br />
2. Use a micropipette tip to pick a single putative colony off a plate. Insert the tip into the PCR mixture and pipette up and down.<br />
<br />
3. Reserve bacteria from each PCR mixture by removing 1 ul and placing into 100 ul of LB + Amp in a labeled tube.<br />
<br />
4. Conduct PCR according to the following thermal profile:<br />
<br />
*a. 94 C 10 minutes<br />
*b. 20 cycles of: 94 C 15 seconds, 46 C 15 seconds, 74 C 30 seconds<br />
*c. 74 C 5 minutes<br />
<br />
5. Add 5 ul 5X loading buffer and run 14 ul on polyacrylamide or agarose gel.<br />
<br />
6. Grow desired clones from reserved bacteria for use in plasmid preps.</div>Macampbellhttp://2007.igem.org/wiki/index.php/Splitting_Genes_for_HPPSplitting Genes for HPP2007-06-15T16:24:31Z<p>Macampbell: </p>
<hr />
<div>We have selected 4 genes to split. We will use our [http://gcat.davidson.edu/iGEM07/genesplitter.html online gene splitting tool] to choose the PCR primers. Davidson will produce these 4 split genes and test each one. <br />
<br />
# Red Fluorescent Protein<br />
# Kanamycin Nucleotidyltransferase <br />
# Cre Recombinase<br />
# Chloramphenicol Acetyl Transferase<br />
<br />
<br />
We will also produce two constructs for tetsing promoters. MWSU will produec (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes. <br />
<br />
Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and lack the backwards promotion we have detected with Part: [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. We will test out the modified promoter [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] which is reported to have good repression and strong induction. We may still introduce the UV5 double mutation to enhance transcription and compare with R0010.<br />
<br />
MWSU is also going to produce backwards LacIq to put upstream of pLac [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. The purpose of this is to have more LacIq in the cytoplasm at all times, regardless of ITPG status. <br />
<br />
MWSU is also testing two of the [http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bio372.html one-time-flippable HixC sites] produced by Bruce spring 2007. <br />
<br />
MWSU is going to produce a new Hin-LVA expression plasmid that has ColE1 ori and uses Tet as the selectable marker. <br />
<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br><br />
----<br />
'''DsRed - Red Fluorescent Protein'''<br />
<br />
We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 ''Discosoma sp.'']), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.<br />
<br />
Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.<br />
<br />
[[Image:Rfp hix insertion point.jpg]]<br />
<br><br />
----<br />
<br />
'''Kanamycin Nucleotidyltransferase'''<br />
<br />
One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA.[http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1] <br />
Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimmer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that: <br />
<br />
-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]<br />
<br />
-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-We did not consider any Amino Acids near the N or C terminus <br />
<br />
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)<br />
<br />
[[Image:KNTase hix cut.png]]<br />
<br />
The yellow bands at the top and bottom of the molecule denotes hix site insertion<br />
<br />
We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.<br />
<br><br />
----<br />
<br />
'''Chloramphenicol Acetyltransferase'''<br />
<br><br />
----<br />
<br />
'''Cre Recombinase'''<br />
<br />
[[Image:Cre recombinase monomer.png]]<br />
<br />
[[Image:Cre recombinase tetramer.png]]<br />
<br />
[http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT?CRETRY=1&SRETRY=0].<br />
<br />
<br><br />
----<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br></div>Macampbellhttp://2007.igem.org/wiki/index.php/Splitting_Genes_for_HPPSplitting Genes for HPP2007-06-14T19:10:11Z<p>Macampbell: </p>
<hr />
<div>We have selected 4 genes to split. We will use our [http://gcat.davidson.edu/iGEM07/genesplitter.html online gene splitting tool] to choose the PCR primers. Davidson will produce these 4 split genes and test each one. <br />
<br />
# Red Fluorescent Protein<br />
# Kanamycin Nucleotidyltransferase <br />
# Cre Recombinase<br />
# Chloramphenicol Acetyl Transferase<br />
<br />
<br />
We will also produce two constructs for tetsing promoters. MWSU will produec (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes. <br />
<br />
Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and lack the backwards promotion we have detected with Part: [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. We will test out the modified promoter [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] which is reported to have good repression and strong induction. We may still introduce the UV5 double mutation to enhance transcription and compare with R0010.<br />
<br />
MWSU is also going to produce backwards LacIq to put upstream of pLac [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. The purpose of this is to have more LacIq in the cytoplasm at all times, regardless of ITPG status. <br />
<br />
MWSU is also testing two of the [http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bio372.html one-time-flippable HixC sites] produced by Bruce spring 2007. <br />
<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br><br />
----<br />
'''DsRed - Red Fluorescent Protein'''<br />
<br />
We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 ''Discosoma sp.'']), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.<br />
<br />
Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.<br />
<br />
[[Image:Rfp hix insertion point.jpg]]<br />
<br><br />
----<br />
<br />
'''Kanamycin Nucleotidyltransferase'''<br />
<br />
One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA.[http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1] <br />
Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimmer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that: <br />
<br />
-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]<br />
<br />
-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-We did not consider any Amino Acids near the N or C terminus <br />
<br />
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)<br />
<br />
[[Image:KNTase hix cut.png]]<br />
<br />
The yellow bands at the top and bottom of the molecule denotes hix site insertion<br />
<br />
We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.<br />
<br><br />
----<br />
<br />
'''Chloramphenicol Acetyltransferase'''<br />
<br><br />
----<br />
<br />
'''Cre Recombinase'''<br />
<br />
[[Image:Cre recombinase monomer.png]]<br />
<br />
[[Image:Cre recombinase tetramer.png]]<br />
<br />
[http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT?CRETRY=1&SRETRY=0].<br />
<br />
<br><br />
----<br />
[https://2007.igem.org/Davidson_Missouri_W return to DMW main page]<br />
<br></div>Macampbellhttp://2007.igem.org/wiki/index.php/Splitting_Genes_for_HPPSplitting Genes for HPP2007-06-14T19:08:17Z<p>Macampbell: </p>
<hr />
<div>We have selected 4 genes to split. We will use our [http://gcat.davidson.edu/iGEM07/genesplitter.html online gene splitting tool] to choose the PCR primers. Davidson will produce these 4 split genes and test each one. <br />
<br />
# Red Fluorescent Protein<br />
# Kanamycin Nucleotidyltransferase <br />
# Cre Recombinase<br />
# Chloramphenicol Acetyl Transferase<br />
<br />
<br />
We will also produce two constructs for tetsing promoters. MWSU will produec (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes. <br />
<br />
Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and lack the backwards promotion we have detected with Part: [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. We will test out the modified promoter [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] which is reported to have good repression and strong induction. We may still introduce the UV5 double mutation to enhance transcription and compare with R0010.<br />
<br />
MWSU is also going to produce backwards LacIq to put upstream of pLac [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. The purpose of this is to have more LacIq in the cytoplasm at all times, regardless of ITPG status. <br />
<br />
MWSU is also testing two of the [http://www.bio.davidson.edu/Courses/genomics/2006/henschen/Bio372.html one-time-flippable HixC sites] produced by Bruce spring 2007. <br />
<br />
<br><br />
----<br />
'''DsRed - Red Fluorescent Protein'''<br />
<br />
We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 ''Discosoma sp.'']), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.<br />
<br />
Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.<br />
<br />
[[Image:Rfp hix insertion point.jpg]]<br />
<br><br />
----<br />
<br />
'''Kanamycin Nucleotidyltransferase'''<br />
<br />
One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA.[http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1] <br />
Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimmer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that: <br />
<br />
-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]<br />
<br />
-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-We did not consider any Amino Acids near the N or C terminus <br />
<br />
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)<br />
<br />
[[Image:KNTase hix cut.png]]<br />
<br />
The yellow bands at the top and bottom of the molecule denotes hix site insertion<br />
<br />
We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.<br />
<br><br />
----<br />
<br />
'''Chloramphenicol Acetyltransferase'''<br />
<br><br />
----<br />
<br />
'''Cre Recombinase'''<br />
<br />
[[Image:Cre recombinase monomer.png]]<br />
<br />
[[Image:Cre recombinase tetramer.png]]<br />
<br />
[http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT?CRETRY=1&SRETRY=0].<br />
<br />
<br><br />
----</div>Macampbellhttp://2007.igem.org/wiki/index.php/Splitting_Genes_for_HPPSplitting Genes for HPP2007-06-14T19:07:03Z<p>Macampbell: </p>
<hr />
<div>We have selected 4 genes to split. We will use our [http://gcat.davidson.edu/iGEM07/genesplitter.html online gene splitting tool] to choose the PCR primers. Davidson will produce these 4 split genes and test each one. <br />
<br />
# Red Fluorescent Protein<br />
# Kanamycin Nucleotidyltransferase <br />
# Cre Recombinase<br />
# Chloramphenicol Acetyl Transferase<br />
<br />
<br />
We will also produce two constructs for tetsing promoters. MWSU will produec (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes. <br />
<br />
Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and lack the backwards promotion we have detected with Part: [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. We will test out the modified promoter [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] which is reported to have good repression and strong induction. We may still introduce the UV5 double mutation to enhance transcription and compare with R0010.<br />
<br />
MWSU is also going to produce backwards LacIq to put upstream of pLac [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. The purpose of this is to have more LacIq in the cytoplasm at all times, regardless of ITPG status. <br />
<br />
<br><br />
----<br />
'''DsRed - Red Fluorescent Protein'''<br />
<br />
We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 ''Discosoma sp.'']), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.<br />
<br />
Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.<br />
<br />
[[Image:Rfp hix insertion point.jpg]]<br />
<br><br />
----<br />
<br />
'''Kanamycin Nucleotidyltransferase'''<br />
<br />
One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA.[http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1] <br />
Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimmer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that: <br />
<br />
-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]<br />
<br />
-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-We did not consider any Amino Acids near the N or C terminus <br />
<br />
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)<br />
<br />
[[Image:KNTase hix cut.png]]<br />
<br />
The yellow bands at the top and bottom of the molecule denotes hix site insertion<br />
<br />
We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.<br />
<br><br />
----<br />
<br />
'''Chloramphenicol Acetyltransferase'''<br />
<br><br />
----<br />
<br />
'''Cre Recombinase'''<br />
<br />
[[Image:Cre recombinase monomer.png]]<br />
<br />
[[Image:Cre recombinase tetramer.png]]<br />
<br />
[http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT?CRETRY=1&SRETRY=0].<br />
<br />
<br><br />
----</div>Macampbellhttp://2007.igem.org/wiki/index.php/Splitting_Genes_for_HPPSplitting Genes for HPP2007-06-14T19:06:25Z<p>Macampbell: </p>
<hr />
<div>We have selected 4 genes to split. We will use our [http://gcat.davidson.edu/iGEM07/genesplitter.html online gene splitting tool] to choose the PCR primers. Davidson will produce these 4 split genes and test each one. <br />
<br />
# Red Fluorescent Protein<br />
# Kanamycin Nucleotidyltransferase <br />
# Cre Recombinase<br />
# Chloramphenicol Acetyl Transferase<br />
<br />
<br />
We will also produce two constructs for tetsing promoters. MWSU will produec (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes. <br />
<br />
Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and lack the backwards promotion we have detected with Part: [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. We will test out the modified promoter [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] which is reported to have good repression and strong induction. We may still introduce the UV5 double mutation to enhance transcription and compare with R0010.<br />
<br />
MWSU is also going to produce backwards LacIq to put upstream of pLac [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. <br />
<br />
<br><br />
----<br />
'''DsRed - Red Fluorescent Protein'''<br />
<br />
We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 ''Discosoma sp.'']), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.<br />
<br />
Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.<br />
<br />
[[Image:Rfp hix insertion point.jpg]]<br />
<br><br />
----<br />
<br />
'''Kanamycin Nucleotidyltransferase'''<br />
<br />
One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA.[http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1] <br />
Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimmer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that: <br />
<br />
-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]<br />
<br />
-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
<br />
-We did not consider any Amino Acids near the N or C terminus <br />
<br />
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)<br />
<br />
[[Image:KNTase hix cut.png]]<br />
<br />
The yellow bands at the top and bottom of the molecule denotes hix site insertion<br />
<br />
We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.<br />
<br><br />
----<br />
<br />
'''Chloramphenicol Acetyltransferase'''<br />
<br><br />
----<br />
<br />
'''Cre Recombinase'''<br />
<br />
[[Image:Cre recombinase monomer.png]]<br />
<br />
[[Image:Cre recombinase tetramer.png]]<br />
<br />
[http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT?CRETRY=1&SRETRY=0].<br />
<br />
<br><br />
----</div>Macampbellhttp://2007.igem.org/wiki/index.php/Splitting_Genes_for_HPPSplitting Genes for HPP2007-06-14T18:27:30Z<p>Macampbell: </p>
<hr />
<div>We have selected 4 genes to split. We will use our [http://gcat.davidson.edu/iGEM07/genesplitter.html online gene splitting tool] to choose the PCR primers. Davidson will produce these 4 split genes and test each one. <br />
<br />
# Red Fluorescent Protein<br />
# Kanamycin Nucleotidyltransferase <br />
# Cre Recombinase<br />
# Chloramphenicol Acetyl Transferase<br />
<br />
<br />
We will also produce two constructs for tetsing promoters. MWSU will produec (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes. <br />
<br />
Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and lack the backwards promotion we have detected with Part: [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. We need to consider Part: [http://partsregistry.org/Part:BBa_I14032 BBa_I14032] first, though. <br />
<br />
MWSU is also going to produce backwards LacIq to put upstream of pLac [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. <br />
<br />
<br><br />
----<br />
'''DsRed - Red Fluorescent Protein'''<br />
<br />
We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 ''Discosoma sp.'']), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.<br />
<br />
Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.<br />
<br />
[[Image:Rfp hix insertion point.jpg]]<br />
<br><br />
----<br />
<br />
'''Kanamycin Nucleotidyltransferase'''<br />
<br />
One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA.[http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1] <br />
Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimmer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that: <br />
<br />
-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
<br />
-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
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-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]<br />
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-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
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-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
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-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]<br />
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-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
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-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]<br />
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-We did not consider any Amino Acids near the N or C terminus <br />
<br />
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)<br />
<br />
[[Image:KNTase hix cut.png]]<br />
<br />
The yellow bands at the top and bottom of the molecule denotes hix site insertion<br />
<br />
We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.<br />
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<br />
'''Chloramphenicol Acetyltransferase'''<br />
<br><br />
----<br />
<br />
'''Cre Recombinase'''<br />
<br />
[[Image:Cre recombinase monomer.png]]<br />
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[[Image:Cre recombinase tetramer.png]]<br />
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[http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT?CRETRY=1&SRETRY=0].<br />
<br />
<br><br />
----</div>Macampbell