Team:Heidelberg/Notebook/Sensing Group/Notebook/4thweek

From 2008.igem.org

(Difference between revisions)
(→Monday, 08/25/2008)
(→Thursday, 08/28/2008)
 
(16 intermediate revisions not shown)
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<html>
<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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#bodyContent { padding: 10px auto; width: 910px; margin: auto; clear: none; }
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li:hover works in IE7 and FF2.
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a:hover works in IE6 and FF2.
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a:hover breaks li:hover in FF2.
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*/
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div.MenuBar ul li:hover ul.DropDownMenu li ul.SideMenu,
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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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div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,
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 +
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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. */
 +
}
 +
/*----------------------------------------------------------------------------- Browser Specific */
 +
* html div.MenuBar ul li a {
 +
  float: left; /* Required for IE55 and IE6.
 +
                            Breaks O9.
 +
                            Hidden (* html) from non-IE browsers. */
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* html ul.DropDownMenu li a:hover {
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  cursor: hand; /* Required for IE55.
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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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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 {
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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,
 +
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
 +
  www.CesarDaniel.info
 +
/*-------------------------------------------------------------------------------------- General */
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  font: 13px arial, helvetica, sans-serif; /* Required for IE55. */
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div.MenuBar a,
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div.MenuBar ul li:hover ul.DropDownMenu li a,
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div.MenuBar ul li a:hover ul.DropDownMenu li a,
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div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,
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div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a {
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div.MenuBar ul li:hover a,
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div.MenuBar ul li a:hover,
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div.MenuBar ul li:hover ul.DropDownMenu li:hover a,
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div.MenuBar ul li a:hover ul.DropDownMenu li a:hover,
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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:hover ul.DropDownMenu,
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div.MenuBar ul li a:hover ul.DropDownMenu,
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div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,
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/*--------------------------------------------------------------------------------------- Widths */
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/*
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Menu Bar 1
 +
Drop-Down Menu #2
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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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/*...............................................................................................*/
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/*
 +
Menu Bar 1
 +
Drop-Down Menu #2
 +
Side Menu #1
 +
*/
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div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1,
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                            Keep it sync with the drop-down menu width. */
 +
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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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div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1 li a,
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 +
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 +
/*...............................................................................................*/
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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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                            Keep it sync with the drop-down menu width. */
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 +
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2,
 +
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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div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2 li a {
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/*...............................................................................................*/
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/*
 +
Menu Bar 1
 +
Drop-Down Menu #2
 +
Side Menu #3
 +
*/
 +
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 {
 +
  left: 15.5em  !important; /* 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. */
 +
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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 {
 +
  width: 10em; /* Side menu width. */
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}
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/*...............................................................................................*/
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 +
</style>
 +
 
<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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* sequentiell and simultaneous digestion of pDk48 NcoI/NdeI and NcoI/KpnI
* sequentiell and simultaneous digestion of pDk48 NcoI/NdeI and NcoI/KpnI
--> not successful
--> not successful
-
* preparation of O/N culture of pDK48 transformed cells
+
* preparation of O/N culture for pDK48 cells
== Tuesday, 08/26/2008 ==
== Tuesday, 08/26/2008 ==
-
* digestion of pDK48 with XbaI and PstI to check wether the vector is the right one
 
 +
=== Cloning ===
 +
[[Image:HD 080826-pDK48 digestions.png|right|thumb|200px|Digestion of pDK48 with xbaI and PstI yield expected results]]
 +
* test digestion of pDK48 with XbaI (NEBuffer 2 + BSA) and PstI (NeBuffer 3 + BSA) to check wehther the vector is the right one
 +
* digestion of Fusion-1 (from 08/21/2008) with NcoI/NdeI, with subsequent gel extraction
 +
* Ligation of Fusion-1 fragment and pDk48 plasmid and transformation into DH5α competent cells
 +
 +
=== LuxS Test ===
 +
'''Material''': 500 mM IPTG-stock (A), 10 mM IPTG-stock (B)
 +
# negative control: just 1 ml cell suspension
 +
# 10 µM IPTG induced (1 µl stock B + 999 µl cell suspension)
 +
# 20 µM IPTG induced (2 µl stock B + 998 µl cell suspension)
 +
# 50 µM IPTG induced (5 µl stock B + 995 µl cell suspension)
 +
# 100 µM IPTG induced (10 µl stock B + 990 µl cell suspension)
 +
# 500 µM IPTG induced (1 µl stock A + 999 µl cell suspension)
 +
# 1 mM IPTG induced (2 µl stock A + 998 µl celll suspension)
 +
 +
Measurement was done like described in the [[Team:Heidelberg/Notebook/material | Materials & Methods]] section
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== Wednesday, 08/27/2008 ==
== Wednesday, 08/27/2008 ==
 +
* PCR of LuxQ from ''V. harveyi'' with Phusion Mastermix
 +
** PCR cycle: 5min @ 98°C || 10s @ 98°C | 30s @ 50°C | 1min @ 72°C || 5min @ 72°C | 4°C hold (30 cycles)
 +
** NO PCR product
 +
* preparation of pTrc99α and pDK48 O/N cultures for Maxipreps
== Thursday, 08/28/2008 ==
== Thursday, 08/28/2008 ==
 +
* Maxiprep of pDK48 and pTrc99α (product eluted in TB buffer, not water!!)
 +
* Digestion of LuxQ-Tar fusion (A,B) and LuxQ 1-10 with the follwing enzymes:
 +
*Q(1-10):
 +
*# XbaI (NEBuffer 2 + BSA) --> if insert: 736 + 1204 bp band; if no insert: 4176 bp band
 +
*# EcoRV (NEBuffer 3 + BSA) --> if insert: 5074 bp + 866 band; if no insert: 4176 bp
 +
*F1:
 +
*# BamHI (NEBuffer 3 + BSA) --> if insert: 6101 bp band; if no insert: 5768 + 833 bp band
 +
*# PstI (NEBuffer 3 + BSA --> if insert: 6101 bp; if no insert: 5768 + 833 bp band
 +
--> No result
== Friday, 08/29/2008 ==
== Friday, 08/29/2008 ==
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/5thweek | Go to 5th week]]
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/5thweek | Go to 5th week]]

Latest revision as of 14:03, 29 October 2008

Back to the overview

Contents

Monday, 08/25/2008

  • sequentiell and simultaneous digestion of pDk48 NcoI/NdeI and NcoI/KpnI

--> not successful

  • preparation of O/N culture for pDK48 cells

Tuesday, 08/26/2008

Cloning

Digestion of pDK48 with xbaI and PstI yield expected results
  • test digestion of pDK48 with XbaI (NEBuffer 2 + BSA) and PstI (NeBuffer 3 + BSA) to check wehther the vector is the right one
  • digestion of Fusion-1 (from 08/21/2008) with NcoI/NdeI, with subsequent gel extraction
  • Ligation of Fusion-1 fragment and pDk48 plasmid and transformation into DH5α competent cells

LuxS Test

Material: 500 mM IPTG-stock (A), 10 mM IPTG-stock (B)

  1. negative control: just 1 ml cell suspension
  2. 10 µM IPTG induced (1 µl stock B + 999 µl cell suspension)
  3. 20 µM IPTG induced (2 µl stock B + 998 µl cell suspension)
  4. 50 µM IPTG induced (5 µl stock B + 995 µl cell suspension)
  5. 100 µM IPTG induced (10 µl stock B + 990 µl cell suspension)
  6. 500 µM IPTG induced (1 µl stock A + 999 µl cell suspension)
  7. 1 mM IPTG induced (2 µl stock A + 998 µl celll suspension)

Measurement was done like described in the Materials & Methods section

Wednesday, 08/27/2008

  • PCR of LuxQ from V. harveyi with Phusion Mastermix
    • PCR cycle: 5min @ 98°C || 10s @ 98°C | 30s @ 50°C | 1min @ 72°C || 5min @ 72°C | 4°C hold (30 cycles)
    • NO PCR product
  • preparation of pTrc99α and pDK48 O/N cultures for Maxipreps

Thursday, 08/28/2008

  • Maxiprep of pDK48 and pTrc99α (product eluted in TB buffer, not water!!)
  • Digestion of LuxQ-Tar fusion (A,B) and LuxQ 1-10 with the follwing enzymes:
  • Q(1-10):
    1. XbaI (NEBuffer 2 + BSA) --> if insert: 736 + 1204 bp band; if no insert: 4176 bp band
    2. EcoRV (NEBuffer 3 + BSA) --> if insert: 5074 bp + 866 band; if no insert: 4176 bp
  • F1:
    1. BamHI (NEBuffer 3 + BSA) --> if insert: 6101 bp band; if no insert: 5768 + 833 bp band
    2. PstI (NEBuffer 3 + BSA --> if insert: 6101 bp; if no insert: 5768 + 833 bp band

--> No result

Friday, 08/29/2008

Go to 5th week

Retrieved from "http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook/4thweek"