Team:UT Dallas/results

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<b>Promoter Evaluation (Sugar sensor)</b><br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We began by testing the relative strength of the promoters we added to the registry: pFru (BBa_K1214007) and pScr (BBa_K1214006). Our promoters and the medium constitutive promoter (BBa_K823014) were ligated with RBS-GFP, which served as our reporter molecule, and subsequently transformed into our chassis organism. The experiment we ran used glycerol stocks from this transformation, inoculated in 5mL of chloramphenicol broth overnight. The resulting cell cultures were then used for Flow cytometry analysis and microscope imaging.<br><br>
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<center><img src="https://static.igem.org/mediawiki/parts/6/6f/FlowPromoters.png"/><br><br>
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CP-GFP: Bright field (left) versus GFP Expression at 395nm (right)<br>
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<img src="http://i826.photobucket.com/albums/zz188/lana_khazma/fancy%20stuff/1_zps40df5709.png" border="0" alt=" photo 1_zps40df5709.png"/><img src="http://i826.photobucket.com/albums/zz188/lana_khazma/fancy%20stuff/2_zpsb7ccdfae.png" border="0" alt=" photo 2_zpsb7ccdfae.png"/><br>
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pScr-GFP: Bright field (left) versus GFP Expression at 395nm (right)<br>
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<img src="http://i826.photobucket.com/albums/zz188/lana_khazma/3_zpsebbfb6cc.png" border="0" alt=" photo 3_zpsebbfb6cc.png"/><img src="http://i826.photobucket.com/albums/zz188/lana_khazma/fancy%20stuff/4_zpse136b2d5.png" border="0" alt=" photo 4_zpse136b2d5.png"/><br>
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pFru-GFP: Bright field (left) versus GFP Expression at 395nm (right)<br>
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<img src="http://i826.photobucket.com/albums/zz188/lana_khazma/5_zpsa845b24d.png" border="0" alt=" photo 5_zpsa845b24d.png"/><img src="http://i826.photobucket.com/albums/zz188/lana_khazma/6_zps8f6d75ed.png" border="0" alt=" photo 6_zps8f6d75ed.png"/>
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<br><br></center>
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<b>Simulations (Com Sensor)</b><br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We used a modeling program called gro to simulate our Com system (http://depts.washington.edu/soslab/gro/). We have set up two populations, one for S.Mutans and one for E.Coli. The E.Coli release CSP, a quorum signaling peptide unique to S.Mutans, which triggers the S.Mutans to undergo apoptosis. As the simulation progresses, the S.Mutans cells will die off as the E.Coli constantly secrete more CSP.<br><br>
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<a rel='lightbox' title='' href='http://i826.photobucket.com/albums/zz188/lana_khazma/gro1_zps1edca3cc.png'>
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<img src="http://i826.photobucket.com/albums/zz188/lana_khazma/gro1_zps1edca3cc.png" height="400"/></a><br><br>
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<a rel='lightbox' title='' href='http://i826.photobucket.com/albums/zz188/lana_khazma/gro2_zps8b49cb60.png'>
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<img src="http://i826.photobucket.com/albums/zz188/lana_khazma/gro2_zps8b49cb60.png" height="400"/></a><br><br>
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<a rel='lightbox' title='' href='http://i826.photobucket.com/albums/zz188/lana_khazma/gro3_zps13098071.png'>
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<img src="http://i826.photobucket.com/albums/zz188/lana_khazma/gro3_zps13098071.png" height="400"/></a><br><br>
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<a rel='lightbox' title='' href='http://i826.photobucket.com/albums/zz188/lana_khazma/gro4_zps6809e675.png'>
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<img src="http://i826.photobucket.com/albums/zz188/lana_khazma/gro4_zps6809e675.png" height="400"/></a><br><br>
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<b>Norspermidine (NspC)</b><br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In order to test the ability of norspermidine on the growth of biofilms in s. mutans we designed an experiment which consisted of several different plates. All plates were spread with s. mutans cells that had been growing in broth. We then spread some of these plates with e. coli cells which contained the plasmid we had created. Finally, we used a centrifuge to spin down a tube of our e. coli in broth. The remaining broth was then spread on some of our plates with the s. mutans. Below is a picture of two of our plates. The plate that was only spread with s. mutans contained a smooth "lawn" of s. mutans. The plates which had been spread with norspermidine-rich broth still contained many colonies, but the colonies did not form a "lawn".
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<a rel='lightbox' title='' href='http://i826.photobucket.com/albums/zz188/lana_khazma/nspcplates_zpsc0bb63e1.jpg'>
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<img src="http://i826.photobucket.com/albums/zz188/lana_khazma/nspcplates_zpsc0bb63e1.jpg" height="400"/></a><br><br>
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<script type="text/javascript">
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$('#gallery a').lightBox({fixedNavigation:true});
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$("a[rel='lightbox']").lightBox();
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</script>

Latest revision as of 16:35, 25 October 2013

Promoter Evaluation (Sugar sensor)
     We began by testing the relative strength of the promoters we added to the registry: pFru (BBa_K1214007) and pScr (BBa_K1214006). Our promoters and the medium constitutive promoter (BBa_K823014) were ligated with RBS-GFP, which served as our reporter molecule, and subsequently transformed into our chassis organism. The experiment we ran used glycerol stocks from this transformation, inoculated in 5mL of chloramphenicol broth overnight. The resulting cell cultures were then used for Flow cytometry analysis and microscope imaging.

<img src="FlowPromoters.png"/>

CP-GFP: Bright field (left) versus GFP Expression at 395nm (right)
<img src="http://i826.photobucket.com/albums/zz188/lana_khazma/fancy%20stuff/1_zps40df5709.png" border="0" alt=" photo 1_zps40df5709.png"/><img src="http://i826.photobucket.com/albums/zz188/lana_khazma/fancy%20stuff/2_zpsb7ccdfae.png" border="0" alt=" photo 2_zpsb7ccdfae.png"/>

pScr-GFP: Bright field (left) versus GFP Expression at 395nm (right)
<img src="http://i826.photobucket.com/albums/zz188/lana_khazma/3_zpsebbfb6cc.png" border="0" alt=" photo 3_zpsebbfb6cc.png"/><img src="http://i826.photobucket.com/albums/zz188/lana_khazma/fancy%20stuff/4_zpse136b2d5.png" border="0" alt=" photo 4_zpse136b2d5.png"/>

pFru-GFP: Bright field (left) versus GFP Expression at 395nm (right)
<img src="http://i826.photobucket.com/albums/zz188/lana_khazma/5_zpsa845b24d.png" border="0" alt=" photo 5_zpsa845b24d.png"/><img src="http://i826.photobucket.com/albums/zz188/lana_khazma/6_zps8f6d75ed.png" border="0" alt=" photo 6_zps8f6d75ed.png"/>





Simulations (Com Sensor)

     We used a modeling program called gro to simulate our Com system (http://depts.washington.edu/soslab/gro/). We have set up two populations, one for S.Mutans and one for E.Coli. The E.Coli release CSP, a quorum signaling peptide unique to S.Mutans, which triggers the S.Mutans to undergo apoptosis. As the simulation progresses, the S.Mutans cells will die off as the E.Coli constantly secrete more CSP.

<a rel='lightbox' title= href='http://i826.photobucket.com/albums/zz188/lana_khazma/gro1_zps1edca3cc.png'> <img src="http://i826.photobucket.com/albums/zz188/lana_khazma/gro1_zps1edca3cc.png" height="400"/></a>

<a rel='lightbox' title= href='http://i826.photobucket.com/albums/zz188/lana_khazma/gro2_zps8b49cb60.png'> <img src="http://i826.photobucket.com/albums/zz188/lana_khazma/gro2_zps8b49cb60.png" height="400"/></a>

<a rel='lightbox' title= href='http://i826.photobucket.com/albums/zz188/lana_khazma/gro3_zps13098071.png'> <img src="http://i826.photobucket.com/albums/zz188/lana_khazma/gro3_zps13098071.png" height="400"/></a>

<a rel='lightbox' title= href='http://i826.photobucket.com/albums/zz188/lana_khazma/gro4_zps6809e675.png'> <img src="http://i826.photobucket.com/albums/zz188/lana_khazma/gro4_zps6809e675.png" height="400"/></a>


Norspermidine (NspC)
     In order to test the ability of norspermidine on the growth of biofilms in s. mutans we designed an experiment which consisted of several different plates. All plates were spread with s. mutans cells that had been growing in broth. We then spread some of these plates with e. coli cells which contained the plasmid we had created. Finally, we used a centrifuge to spin down a tube of our e. coli in broth. The remaining broth was then spread on some of our plates with the s. mutans. Below is a picture of two of our plates. The plate that was only spread with s. mutans contained a smooth "lawn" of s. mutans. The plates which had been spread with norspermidine-rich broth still contained many colonies, but the colonies did not form a "lawn".


<a rel='lightbox' title= href='http://i826.photobucket.com/albums/zz188/lana_khazma/nspcplates_zpsc0bb63e1.jpg'> <img src="http://i826.photobucket.com/albums/zz188/lana_khazma/nspcplates_zpsc0bb63e1.jpg" height="400"/></a>



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