Team:UT Dallas/Parts

From 2013.igem.org

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<div id="bottom-panel">
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<table border='1' cellspacing='1'>
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<tr>
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<td valign='top'><b>Name</b></td>
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<td valign='top'><b>Type</b></td>
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<td valign='top'><b>Description</b></td>
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<td valign='top'><b>Designer</b></td>
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<td valign='top'><b>Length</b></td>
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<td valign='top'><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1214000">BBa_K1214000</a></td>
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<td valign='top'>Signalling</td>
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<td valign='top'>ComC, Codes for Competence Signalling Peptide(CSP) in Streptococcus Mutans, Works with ComB and ComA</td>
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<td valign='top'>Alec Spinhirne</td>
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<td valign='top'>139</td>
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</tr>
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<tr>
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<td valign='top'><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1214005">BBa_K1214005</a></td>
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<td valign='top'>Regulatory</td>
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<td valign='top'>ScrR, Sucrose Repressor. Represses the sucrose operon promoter pScr. Inactivated by sucrose </td>
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<td valign='top'>Alec Spinhirne</td>
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<td valign='top'>963</td>
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</tr>
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<tr>
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<td valign='top'><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1214006">BBa_K1214006</a></td>
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<td valign='top'>Regulatory</td>
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<td valign='top'>pScr, Sucrose operon promoter. Regulated by sucrose operon repressor, ScrR</td>
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<td valign='top'>Alec Spinhirne</td>
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<td valign='top'>92</td>
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</tr>
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<tr>
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<td valign='top'><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1214007">BBa_K1214007</a></td>
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<td valign='top'>Regulatory</td>
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<td valign='top'>pFru, Fructose operon promoter. Regulated by fructose operon repressor FruR</td>
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<td valign='top'>Alec Spinhirne</td>
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<td valign='top'>180</td>
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</tr>
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<tr>
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<td valign='top'><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1214008">BBa_K1214008</a></td>
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<td valign='top'>Coding</td>
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<td valign='top'>FruR, Fructose operon repressor, Represses fructose operon promoter pFru.</td>
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<td valign='top'>Alec Spinhirne</td>
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<td valign='top'>1007</td>
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</tr>
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<tr>
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<td valign='top'><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1214009">BBa_K1214009</a></td>
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<td valign='top'>Coding</td>
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<td valign='top'>Dex, Encodes for dextranase. Membrane-bound protein that binds to dextran.</td>
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<td valign='top'>Alec Spinhirne</td>
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<td valign='top'>2553</td>
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</tr>
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<tr>
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<td valign='top'><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1214010">BBa_K1214010</a></td>
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<td valign='top'>Coding</td>
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<td valign='top'>NspC</td>
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<td valign='top'>Alec Spinhirne</td>
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<td valign='top'>1203</td>
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</tr>
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<tr>
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<td valign='top'><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1214011">BBa_K1214011</a></td>
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<td valign='top'>Composite</td>
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<td valign='top'>pFru-RBS-GFP, Fructose operon promoter with GFP coding sequence downstream.
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Constitutively expresses GFP. Regulated by fructose operon repressor FruR (BBa_K1214008)</td>
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<td valign='top'>Alec Spinhirne</td>
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<td valign='top'>976</td>
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</tr>
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<tr>
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<td valign='top'><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1214012">BBa_K1214012</a></td>
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<td valign='top'>Composite</td>
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<td valign='top'>pScr-RBS-GFP, Sucrose operon promoter with GFP coding sequence downstream. Should constitutively express GFP. Regulated by ScrR (BBa_K1214005)</td>
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<td valign='top'>Alec Spinhirne</td>
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<td valign='top'>888</td>
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</tr>
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<tr>
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<td valign='top'><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1214013">BBa_K1214013</a></td>
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<td valign='top'>Composite</td>
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<td valign='top'>Promoter-RBS-FruR, Constitutive promoter with fructose operon repressor downstream. Should constitutively express FruR (BBa_K1214008) </td>
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<td valign='top'>Alec Spinhirne</td>
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<td valign='top'>1070</td>
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</tr>
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<tr>
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<td valign='top'><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1214014">BBa_K1214014</a></td>
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<td valign='top'>Composite</td>
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<td valign='top'>Promoter-RBS-ScrR, Constitutive promoter with sucrose operon repressor downstream. Should constitutively express ScrR</td>
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<td valign='top'>Alec Spinhirne</td>
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<td valign='top'>&nbsp;</td>
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<td valign='top'><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1214015">BBa_K1214015</a></td>
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#content {
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<td valign='top'>Composite</td>
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<td valign='top'>Promoter-RBS-FruR-TT-pFru-RBS-GFP, Constitutive promoter with fructose operon repressor downstream. Should repress pFru when fructose is not present. When fructose is added to system, GFP should be expressed</td>
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<td valign='top'>Alec Spinhirne</td>
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            <span id="abs-title">PARTS</span>
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== Experiments ==<br>
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----<br>
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A) Promoter Evaluation<br>
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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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[[File:FlowPromoters.png]]<br><br>
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Background Cp-GFP (left) versus GFP Expression at 395nm (right)<br>
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[[File:BFCpGFP.tif]]  [[File:CpGFP.tif]]<br>
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Background pScr-GFP (left) versus GFP Expression at 395nm (right)<br>
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[[File:BFpscrGFP.tif]]  [[File:pScrGfp.tif]]<br>
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Background pFru-GFP (left) versus GFP Expression at 395nm (right)<br>
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[[File:BFpFruGfp.tif]]  [[File:pFruGFP.tif]]
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<br><br>
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B) Repressor Evaluation<br>
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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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        These repressors were transformed to make four systems, with the medium constitutive promoter indicated by Cp: <br>
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### pFru-RBS-GFP-Cp-RBS-ScrR<br>
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### pFru-RBS-GFP-Cp-RBS-Cra<br>
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### pScr-RBS-GFP-Cp-RBS-ScrR<br>
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### pScr-RBS-GFP-Cp-RBS-Cra<br>
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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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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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<br>
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[[File:FlowpScrrepression.png]]<br>
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[[File:FlowpFrurepression.png]]<br>
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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>
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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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Preliminary tests on the inducibility of the repressors was performed but the data was inconclusive.<br>
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            </span>
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<td valign='top'><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1214016">BBa_K1214016</a></td>
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        </td>
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<td valign='top'>Coding</td>
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        <td>
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<td valign='top'>RBS-NspC, Encodes for NspC. Expression should lead to the synthesis of norspermidine, an aid in breaking down biofilms</td>
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          </div>
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<td valign='top'>Alec Spinhirne</td>
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        </td>
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<td valign='top'>1264</td>
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Latest revision as of 00:41, 16 October 2013

Name Type Description Designer Length
BBa_K1214000 Signalling ComC, Codes for Competence Signalling Peptide(CSP) in Streptococcus Mutans, Works with ComB and ComA Alec Spinhirne 139
BBa_K1214005 Regulatory ScrR, Sucrose Repressor. Represses the sucrose operon promoter pScr. Inactivated by sucrose Alec Spinhirne 963
BBa_K1214006 Regulatory pScr, Sucrose operon promoter. Regulated by sucrose operon repressor, ScrR Alec Spinhirne 92
BBa_K1214007 Regulatory pFru, Fructose operon promoter. Regulated by fructose operon repressor FruR Alec Spinhirne 180
BBa_K1214008 Coding FruR, Fructose operon repressor, Represses fructose operon promoter pFru. Alec Spinhirne 1007
BBa_K1214009 Coding Dex, Encodes for dextranase. Membrane-bound protein that binds to dextran. Alec Spinhirne 2553
BBa_K1214010 Coding NspC Alec Spinhirne 1203
BBa_K1214011 Composite pFru-RBS-GFP, Fructose operon promoter with GFP coding sequence downstream. Constitutively expresses GFP. Regulated by fructose operon repressor FruR (BBa_K1214008) Alec Spinhirne 976
BBa_K1214012 Composite pScr-RBS-GFP, Sucrose operon promoter with GFP coding sequence downstream. Should constitutively express GFP. Regulated by ScrR (BBa_K1214005) Alec Spinhirne 888
BBa_K1214013 Composite Promoter-RBS-FruR, Constitutive promoter with fructose operon repressor downstream. Should constitutively express FruR (BBa_K1214008) Alec Spinhirne 1070
BBa_K1214014 Composite Promoter-RBS-ScrR, Constitutive promoter with sucrose operon repressor downstream. Should constitutively express ScrR Alec Spinhirne  
BBa_K1214015 Composite Promoter-RBS-FruR-TT-pFru-RBS-GFP, Constitutive promoter with fructose operon repressor downstream. Should repress pFru when fructose is not present. When fructose is added to system, GFP should be expressed Alec Spinhirne  
BBa_K1214016 Coding RBS-NspC, Encodes for NspC. Expression should lead to the synthesis of norspermidine, an aid in breaking down biofilms Alec Spinhirne 1264

Retrieved from "http://2013.igem.org/Team:UT_Dallas/Parts"