Team:UT Dallas/results

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<span class="title_spans">Results</span><br><br><br>
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<b>Promoter Evaluation (Sugar sensor)</b><br>
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<font size=3><b>Experiments</b><br></font>
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<b> A) &nbsp;&nbsp;&nbsp;Promoter Evaluation</b><br></font>
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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>
&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/b/b7/FlowpFruRepression.png"/><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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Background Cp-GFP (left) versus GFP Expression at 395nm (right)<br>
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CP-GFP: Bright field (left) versus GFP Expression at 395nm (right)<br>
<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>
<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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Background pScr-GFP (left) versus GFP Expression at 395nm (right)<br>
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pScr-GFP: Bright field (left) versus GFP Expression at 395nm (right)<br>
<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>
<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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Background pFru-GFP (left) versus GFP Expression at 395nm (right)<br></font>
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pFru-GFP: Bright field (left) versus GFP Expression at 395nm (right)<br>
<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"/>
<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"/>
<br><br></center>
<br><br></center>
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<b>B) &nbsp;&nbsp;&nbsp;Repressor Evaluation</b><br></font>
 
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Our team submitted two new repressors to the registry that work in conjunction with the aforementioned promoters. These repressors are Cra (BBa_K1214008) and ScrR (BBa_K1214005). In theory, these should be inducible repressors that repress their respective promoters. Cra (also known as FruR) will repress pFru until Fructose is introduced, wherein the repressor will unblock pFru and promotion will continue. The same relationship is expected between ScrR and pScr except that promotion is induced by sucrose in this case.<br>
 
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<blockquote>These repressors were transformed to make four systems, with the medium constitutive promoter indicated by Cp: <br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1. pFru-RBS-GFP-Cp-RBS-ScrR<br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2. pFru-RBS-GFP-Cp-RBS-Cra<br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3. pScr-RBS-GFP-Cp-RBS-ScrR<br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4. pScr-RBS-GFP-Cp-RBS-Cra<br></blockquote>
 
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;These were plated, incubated overnight. Seeing as the plates were from a transformation, not all colonies are guaranteed to contain the completed system. Thus, the GFP expression on these plates would be less than the true GFP expression of the system and they were not used in our characterization.. <br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Colonies were then picked from these plates and inoculated in 5mL of chloramphenicol broth overnight. This inoculation was used to perform Flow cytometry analysis of the repressors. It should be noted that the promotion of the repressors is dictated by Cp more than pFru or pScr, and in conjunction with the aforementioned promoter data it can be seen that pFru may cause greater downstream promotion than Cp but was not used due to conflicts with repression. However, the data obtained shows the expected repression patterns, especially in relation to the specificity of the repressors.
 
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<center><img src="http://i826.photobucket.com/albums/zz188/lana_khazma/fancy%20stuff/pscr_zpsccffdf8a.png" border="0" alt=" photo pscr_zpsccffdf8a.png"/><br>
 
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<img src="http://i826.photobucket.com/albums/zz188/lana_khazma/fancy%20stuff/pFru_zps5ff8d50f.png" border="0" alt=" photo pFru_zps5ff8d50f.png"/></center><br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;These repressors are shown to greatly reduce the GFP seen in the systems without repressors. pFru expression of GFP under the repressor was reduced at least five-fold while pScr expression of GFP was reduced at least ten-fold. <br><br>
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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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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;There is also evidence of the repressor’s specificity shown in the difference between the promoter’s GFP expression depending on whether Cra or ScrR was in the system. The sucrose promoter was repressed approximately four times as much by ScR than by Cra. pFru was repressed roughly 20% more by Cra than by ScrR. This data supports the preexisting claim that the repressors are more specific to their particular promoters. This is important to consider when using our biobricks in a system.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Preliminary tests on the inducibility of the repressors was performed but the data was inconclusive.<br>
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$("a[rel='lightbox']").lightBox();
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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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