Team:Heidelberg/Notebook/Sensing Group/Notebook/11thweek

From 2008.igem.org

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(→Friday, 10/17/2008)
 
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<html>
-
<link rel='stylesheet' href='http://igem-heidelberg.de/fileadmin/Wiki/Heidelberg2.css' type='text/css' />
+
 
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<link rel="stylesheet" href="http://igem-heidelberg.de/fileadmin/Wiki/Menu.css" type="text/css"/>
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<link rel="stylesheet"  href="http://igem-heidelberg.de/fileadmin/Wiki/MenuHD.css" type="text/css"/>
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a:hover breaks li:hover in FF2.
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*/
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div.MenuBar ul li a:hover ul.DropDownMenu li a {
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                            Use MenuTailor.css to customize. */
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ul.DropDownMenu li a span {
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                            Keep it sync with the drop-down menu width.
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                            Use MenuTailor.css to customize. */
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div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,
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                            Use MenuTailor.css to customize. */
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  font: 12px arial, helvetica, sans-serif; /* Required for IE55. */
 +
  left: 13em; /* Places the side menu to the right of the drop-down menu.
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                            Keep it sync with the drop-down menu width.
 +
                            Use MenuTailor.css to customize. */
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}
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div.MenuBar ul li ul.DropDownMenu li ul.SideMenu li a span {
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                            Keep it sync with the drop-down menu width.
 +
                            Use MenuTailor.css to customize. */
 +
}
 +
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* html div.MenuBar ul li a {
 +
  float: left; /* Required for IE55 and IE6.
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                            Breaks O9.
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                            Hidden (* html) from non-IE browsers. */
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* html ul.DropDownMenu li a:hover {
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ul.DropDownMenu li a:hover {
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                      Hidding it (* html) from non-IE browsers breaks IE7!
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* html div.MenuBar a:hover {
 +
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                            Hidden (* html) from non-IE browsers. */
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}
 +
* html div.MenuBar ul li table,
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* html div.MenuBar ul li table td {
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                Hidden (* html) from non-IE browsers. */
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}
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div.MenuBar ul li a:hover ul.DropDownMenu li a {
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  background-color: Menu; /* Side menu unselected items.
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                            Sets the side menu "effective background" color. */
 +
  color: MenuText;
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}
 +
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a:hover,
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div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a:hover {
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/*Script-Free 3-Level Menu 1.2 Tailor
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  www.CesarDaniel.info
 +
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div.MenuBar a,
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div.MenuBar ul li:hover ul.DropDownMenu,
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div.MenuBar ul li a:hover ul.DropDownMenu,
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div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {
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/*
 +
 
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 +
Menu Bar 1
 +
Drop-Down Menu #2
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*/
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div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM4,
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div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM4,
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div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM5,
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div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM5,
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div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM5 li a,
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 +
Menu Bar 1
 +
Drop-Down Menu #2
 +
Side Menu #1
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*/
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div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1,
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div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1,
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 +
Menu Bar 1
 +
Drop-Down Menu #2
 +
Side Menu #2
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div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2,
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div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2,
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div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2 li a,
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Menu Bar 1
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Drop-Down Menu #2
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Side Menu #3
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*/
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 +
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 +
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3,
 +
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3,
 +
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3 li a,
 +
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3 li a {
 +
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/*...............................................................................................*/
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 +
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 +
 
<body>
<body>
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</li>
</li>
<li>
<li>
-
<a href="https://2008.igem.org/Team:Heidelberg/Chemotaxis" style="color: white">Modeling<!--[if gt IE 6]><!--></a><!--<![endif]-->
+
<a href="https://2008.igem.org/Team:Heidelberg/Modeling" style="color: white">Modeling<!--[if gt IE 6]><!--></a><!--<![endif]-->
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
<ul class="DropDownMenu" id="MB1-DDM3">
<ul class="DropDownMenu" id="MB1-DDM3">
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</li>
</li>
<li>
<li>
-
<a href="https://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook" style="color: white">Notebook<!--[if gt IE 6]><!--></a><!--<![endif]-->
+
<a href="https://2008.igem.org/Team:Heidelberg/Notebook/Overview" style="color: white">Notebook<!--[if gt IE 6]><!--></a><!--<![endif]-->
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
<ul class="DropDownMenu" id="MB1-DDM5">
<ul class="DropDownMenu" id="MB1-DDM5">
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</li>
</li>
<li style="width: 160px">
<li style="width: 160px">
-
<a href="https://2008.igem.org/Team:Heidelberg/Project/General_information" style="color: white">Human Practice<!--[if gt IE 6]><!--></a><!--<![endif]-->
+
<a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview" style="color: white">Human Practice<!--[if gt IE 6]><!--></a><!--<![endif]-->
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
<ul class="DropDownMenu" id="MB1-DDM4">
<ul class="DropDownMenu" id="MB1-DDM4">
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                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Surveys"><span><span>Surveys</span></span></a></li>
                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Surveys"><span><span>Surveys</span></span></a></li>
                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Open_Day"><span><span>Open Day</span></span></a></li>
                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Open_Day"><span><span>Open Day</span></span></a></li>
-
                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Nobel_Prize"><span><span>Nobel Prize</span></span></a></li>
+
                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Nobel_Prize"><span><span>Nobel Prize</span></span></a></li>
 +
</ul>
</ul>
<!--[if lte IE 6]></td></tr></table></a><![endif]-->
<!--[if lte IE 6]></td></tr></table></a><![endif]-->
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* Digestion of LuxS with EcoRI/PstI (NEBuffer EcoRI + BSA) and Fusion with BamHI/PstI (NEBuffer 3 + BSA)
* Digestion of LuxS with EcoRI/PstI (NEBuffer EcoRI + BSA) and Fusion with BamHI/PstI (NEBuffer 3 + BSA)
[[Image:HD 081017-LuxS Fusion Digestion.png|center|thumb|400px|Digestion of LuxS with EcoRI/PstI. Positive clones show two bands]]
[[Image:HD 081017-LuxS Fusion Digestion.png|center|thumb|400px|Digestion of LuxS with EcoRI/PstI. Positive clones show two bands]]
-
  Sequencing @ GATC reveal that mutagenesis did not work. Restriction sites still there.
+
  Sequencing @ GATC reveal that mutagenesis did not work. bcause restriction sites are still there.

Latest revision as of 15:40, 27 October 2008

Back to the overview

Contents

Monday, 10/13/2008

  • Miniprep of LuxS_mut_EcoRI and F1-YFP, F2-YFP
  • Digestion of Fusion-YFP with BamHI (NEBuffer3 + BSA)
    • Insert: 5937 bp and 1998 bp
    • No Insert: 7202 bp
  • Digestion of LuxS with EcoRI/NdeI (NEBuffer EcoRI)
    • Insert: 2773 bp and 1862 bp
    • 4635 bp
  • Fusion-PCR for LuxS standardization with Phusion
    • 30s @ 98°C || 10s @ 98°C | 30s @ 55°C | 1min @ 72°C || 5min @ 72°C | 4°C (30 cycles)

Tuesday, 10/14/2008

  • PCR Purification, Digestion of LuxS and pSB2K3 with EcoRI/PstI (EcoRI buffer + BSA). 1h @ 37°C
  • Fusion PCR for LuxS with Phusion and Annealing Temperature Gradient (50-60°C)
Fused LuxS parts for standardization seem positive
  • Transformation of Fusion-YFP into MG1655

Wednesday, 10/15/2008

  • PCR Purification of LuxS_standard and F1-GeneArt, F2-GeneArt
  • Digestion of LuxS and Fusion Constructs with EcoRI/PstI (NEBuffer EcoRI + BSA)
  • Digestion of LuxS with EcoRI/xbaI (NEBuffer2 + BSA) to check for restriction sites
  • Cloning into pSB2K3
  • Test Digestion of LuxS with EcoRI and xbaI to test if mutation was successful
  • Digestion of Fusion1-YFP and Fusion2-YFP constructs with BamHI
LuxS digestions with EcoRI and xbaI to test for successful mutagenesis
Digestion of LuxS to test for mutations and Fusion-YFP with BamHI constructs to confirm insert.


  • Inoculation of 50µl Fusion-YFP O/N culture in 5mL LB. Incubation for 2h @ 37°C. Induction of Fusion-YFP cells with 0.01% Arabinose for 3h. Visualization under microscope
  • Expression of Fusion-Receptor positive for both constructs. Even Localization to the membrane can be seen in some cells.
Fluorescence Image of Fusion1-YFP
Fluorescence Image of Fusion2-YFP


Thursday, 10/16/2008

  • Picked 16 LuxS colonies and Inoculation in LB-Kan
  • Inoculation of UU1250 cells containing Fusion constructs

Friday, 10/17/2008

  • Miniprep of LuxS and Fusion constructs
  • Digestion of LuxS with EcoRI/PstI (NEBuffer EcoRI + BSA) and Fusion with BamHI/PstI (NEBuffer 3 + BSA)
Digestion of LuxS with EcoRI/PstI. Positive clones show two bands
Sequencing @ GATC reveal that mutagenesis did not work. bcause restriction sites are still there.
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