Team:Heidelberg/Notebook/Killing II/10thweek

<html>


<style>
h1.firstHeading { display: none; }

p {text-align: justify;}

a:link { color: #00b0e6; text-decoration: none} 
a:visited { color:#00b0e6; text-decoration: none}
a:hover { color:#f29400; text-decoration: none}
a:active { color:#f29400; text-decoration: none}

#bodyContent { padding: 10px auto; width: 910px; margin: auto; clear: none; }

table#team_members { text-align: justify; border: 0; }
table#team_members h2, table#team_members h3 { clear: both; }


/*-----------------------------------------------------------------------------------------------*/
div.MenuBar ul li ul.DropDownMenu {
  display: none; /* Hides all drop-down menus. */

}
/*
li:hover works in IE7 and FF2.
a:hover works in IE6 and FF2.
a:hover breaks li:hover in FF2.
*/
div.MenuBar ul li:hover ul.DropDownMenu li ul.SideMenu,
div.MenuBar ul li a:hover ul.DropDownMenu li a ul.SideMenu {
  display: none; /* Hides all side menus. */
}
/*------------------------------------------------------------------------------------- Menu Bar */
div.MenuBar {
  font: arial, helvetica, sans-serif;
  height: 30px;
  width: 910px;
  /*width: 100%*/
  margin: 0;
  border-top: 0;
  border-right: 0;
  border-left: 0;
  padding: 0;
  background: black;
  
}
div.MenuBar ul {
  font: arial, helvetica, sans-serif;
  text-align: center;	
  list-style-type: none;
  margin: 0.5em auto;
  border: 0;
  padding: 0;
  background: black;
}
div.MenuBar ul li {
  font: arial, helvetica, sans-serif;
  display: block;
  padding: 0;
  margin: 0;
  font-size: 1.3em;
  float: left;
  background: black;
  text-align: center;
  width: 107px;
  position: relative; /* Sets the positioning context for each drop-down menu. */
}

div.MenuBar ul li a {
  font: arial, helvetica, sans-serif;
  display: block;
  background: black;
  height: 22px; /* Keep height + padding-top + padding-bottom sync with the menu bar height. */
  color: #ffffff;
  padding-top: 4px;
  padding-bottom: 4px;
  padding-left: 1em; /* Sets the left space between top-level items. */
  padding-right: 1em; /* Sets the right space between top-level items. */
  text-decoration: none;
}

/*------------------------------------------------------------------------------ Drop-Down Menus */
div.MenuBar ul li:hover ul.DropDownMenu,
div.MenuBar ul li a:hover ul.DropDownMenu {
  display: block;
  width: 10em; /* Drop-down menu width.
                  Use MenuTailor.css to customize. */
  height: 1em;
  padding: 1px; /* Sets the drop-down menu "effective border" width. */
  position: absolute; 
  top: 23px; /* Places the drop-down menu under the menu bar.
                Keep it sync with the menu bar height. */
  left: 0; /* Aligns the drop-down menu to its top-level item. */
  background-color: black; /* Selected item. */
  color: #FFFFFF;

}
div.MenuBar ul li:hover ul.DropDownMenu li a,
div.MenuBar ul li a:hover ul.DropDownMenu li a {
  width: 10em; /* Keep it sync with the drop-down menu width.
                             Use MenuTailor.css to customize. */
  height: 1em;
  padding-left: 0;
  padding-right: 0;
  background-color: black; /* Selected item. */
  color: #FFFFFF;
}
ul.DropDownMenu li a span {
  display: block;
  padding-left: 0.75em; /* Sets the left space of each drop-down menu item. */
  padding-right: 0.25em; /* Sets the right space of each drop-down menu item. */
  text-align: right; /* Aligns the >> symbol to the right. */
}
ul.DropDownMenu li a span span {
  float: left; /* Aligns the text (back) to the left. */
  font: 12px arial, helvetica, sans-serif; /* Required for IE55. */
  height: 20px;
  color: #FFFFFF;
}
/*----------------------------------------------------------------------------------- Side Menus */
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {
  display: block;
  width: 11em; /* Side menu width.
                  Use MenuTailor.css to customize. */
  padding: 1px; /* Sets the side menu "effective border" width. */
  position: absolute;
  top: -1px; /* Aligns the side menu to its drop-down menu item.
                Keep it sync with the side menu "effective border" width. */
  left: 13em; /* Places the side menu to the right of the drop-down menu.
                            Keep it sync with the drop-down menu width.
                            Use MenuTailor.css to customize. */
}
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a {
  width: 11em; /* Keep it sync with the side menu width.
                             Use MenuTailor.css to customize. */
  font: 12px arial, helvetica, sans-serif; /* Required for IE55. */
  left: 13em; /* Places the side menu to the right of the drop-down menu.
                            Keep it sync with the drop-down menu width.
                            Use MenuTailor.css to customize. */
}
div.MenuBar ul li ul.DropDownMenu li ul.SideMenu li a span {
  padding-left: 1.5em; /* Sets the left space of each side menu item. */
  padding-right: 0.5em; /* Sets the right space of each side menu item. */
  text-align: left;
  font: 12px arial, helvetica, sans-serif; /* Required for IE55. */
  left: 13em; /* Places the side menu to the right of the drop-down menu.
                            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. */
}
* html ul.DropDownMenu li a:hover {
  cursor: hand; /* Required for IE55.
                   Hidden (* html) from non-IE browsers. */
}
ul.DropDownMenu li a:hover {
  cursor: pointer; /* Required for IE6 and IE7.
                      Hidding it (* html) from non-IE browsers breaks IE7! 
}
* html div.MenuBar a:hover {
  text-decoration: none; /* Required for IE55 and IE6.
                            Hidden (* html) from non-IE browsers. */
}
* html div.MenuBar ul li table,
* html div.MenuBar ul li table td {
  border: 0; /* Required for IE55 and IE6.
                Hidden (* html) from non-IE browsers. */
}
/*------------------------------------------------------------------------------- Default Colors */
div.MenuBar {
  background-color: Menu;
  border-bottom: 1px solid ButtonShadow;
}
div.MenuBar a {
  background-color: Menu; /* Top-level unselected items. */
  color: MenuText;
}
div.MenuBar ul li:hover a,
div.MenuBar ul li a:hover {
  color: #ea7f16;
  background-color: Highlight; /* Top-level selected item. */
  color: HighlightText;
}
/*...............................................................................................*/
div.MenuBar ul li:hover ul.DropDownMenu,
div.MenuBar ul li a:hover ul.DropDownMenu {
  background-color: ButtonShadow; /* Sets the drop-down menu "effective border" color. */
}
div.MenuBar ul li:hover ul.DropDownMenu li a,
div.MenuBar ul li a:hover ul.DropDownMenu li a {
  background-color: Menu;  Drop-down menu unselected items.
                             Sets the drop-down menu "effective background" color. */
  color: MenuText;
}
div.MenuBar ul li:hover ul.DropDownMenu li:hover a,
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover {
  background-color: Highlight; /* Drop-down menu selected item. */
  color: HighlightText;
}
/*...............................................................................................*/
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {
  background-color: ButtonShadow; /* Sets the side menu "effective border" color. */
}
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a {
  background-color: Menu; /* Side menu unselected items.
                             Sets the side menu "effective background" color. */
  color: MenuText;
}
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 {
  background-color: Highlight; /* Side menu selected item. */
  color: HighlightText; 
}
/*-----------------------------------------------------------------------------------------------*/
/*Script-Free 3-Level Menu 1.2 Tailor
  www.CesarDaniel.info
/*-------------------------------------------------------------------------------------- General */
body {
  background: white;
  color: black;
  margin: 0;
  border: 0;
  padding: 0;
}


div.MenuBar {
  font: 13px arial, helvetica, sans-serif;
}
div.MenuBar ul {
  font: 13px arial, helvetica, sans-serif; /* Required for IE55. */
}
/*--------------------------------------------------------------------------------------- Colors */
div.MenuBar {
  background-color: black; /* Selected item. */
  color: #FFFFFF;
  border-bottom: 1px solid ButtonShadow;
}
div.MenuBar a,
div.MenuBar ul li:hover ul.DropDownMenu li a,
div.MenuBar ul li a:hover ul.DropDownMenu li a,
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a {
  background-color: black; /* Selected item. */
  color: #FFFFFF;
}
div.MenuBar ul li:hover a,
div.MenuBar ul li a:hover,
div.MenuBar ul li:hover ul.DropDownMenu li:hover a,
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover,
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 {
  background-color: #00b0e6; /* Selected item. */
  color: #FFFFFF;
}
div.MenuBar ul li:hover ul.DropDownMenu,
div.MenuBar ul li a:hover ul.DropDownMenu,
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {
  background-color: ButtonShadow; /* Sets the drop-down and side menus "effective border" color. */
}
/*--------------------------------------------------------------------------------------- Widths */
/*

/*
Menu Bar 1
Drop-Down Menu #2
*/
div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM4,
div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM4,
div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM4 li a,
div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM4 li a {
  width: 11em; /* Drop-down menu width. */
}
div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM5,
div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM5,
div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM5 li a,
div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM5 li a {
  width: 12em; /* Drop-down menu width. */
}

/*...............................................................................................*/
/*
Menu Bar 1
Drop-Down Menu #2
Side Menu #1
*/
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1,
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1 {
  left: 15.5em !important; /* Places the side menu to the right of the drop-down menu.
                            Keep it sync with the drop-down menu width. */
}
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1,
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1,
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1 li a,
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1 li a {
  width: 10em; /* Side menu width. */
}
/*...............................................................................................*/
/*
Menu Bar 1
Drop-Down Menu #2
Side Menu #2
*/
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 {
  left: 15.5em  !important; /* Places the side menu to the right of the drop-down menu.
                            Keep it sync with the drop-down menu width. */
}
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,
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2 li a,
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2 li a {
  width: 10em; /* Side menu width. */
}
/*...............................................................................................*/
/*
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.
                            Keep it sync with the drop-down menu width. */
}
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. */
}
/*...............................................................................................*/

</style>


<body>
<div id="header"><img src="https://static.igem.org/mediawiki/2008/7/78/TeamHeidelbergHeader.jpg" alt="Team Heidelberg" /></div>
<div class='MenuBar' id="navi">
	<ul>
		<li>
			<a href="https://2008.igem.org/Team:Heidelberg" style="color: white">Home
			<!--[if gt IE 6]><!--></a><!--<![endif]-->
			<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
			<!--[if lte IE 6]></td></tr></table></a><![endif]-->
		</li>
		<li>
			<a href="https://2008.igem.org/Team:Heidelberg/Team" style="color: white">Team<!--[if gt IE 6]><!--></a><!--<![endif]-->
			<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
			<ul class="DropDownMenu" id="MB1-DDM6">
			<li><a href="https://2008.igem.org/Team:Heidelberg/Team"><span><span>Overview</span></span></a></li>
			<li><a href="https://2008.igem.org/Team:Heidelberg/Team_Members#Advisors"><span><span>Advisors</span></span></a></li>
			<li><a href="https://2008.igem.org/Team:Heidelberg/Team_Members#Undergraduates"><span><span>Undergraduates</span></span></a></li>
			<li><a href="https://2008.igem.org/Team:Heidelberg/Team#There_are_many_Universities_-_and_there_is_Heidelberg_..."><span><span>University</span></span></a></li>
			<li><a href="https://2008.igem.org/Team:Heidelberg/Team#DKFZ"><span><span>DKFZ</span></span></a></li>
			<li><a href="https://2008.igem.org/Team:Heidelberg/Team#Bioquant"><span><span>BioQuant</span></span></a></li>
			<li><a href="https://2008.igem.org/Team:Heidelberg/BioRegioRN"><span><span>BioRegion Rhein-Neckar</span></span></a></li>
			</ul>
			<!--[if lte IE 6]></td></tr></table></a><![endif]-->
		</li>
		<li>
			<a href="https://2008.igem.org/Team:Heidelberg/Project" style="color: white">Project<!--[if gt IE 6]><!--></a><!--<![endif]-->
			<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
			<ul class="DropDownMenu" id="MB1-DDM1">
			<li><a href="https://2008.igem.org/Team:Heidelberg/Project"><span><span>Overall Project</span></span></a></li>
			<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/material"><span><span>Material & Methods</span></span></a>
			<li><a href="https://2008.igem.org/Team:Heidelberg/Project/Sensing"><span><span>Sensing</span></span></a></li>
			<li><a href="https://2008.igem.org/Team:Heidelberg/Project/Killing_I"><span><span>Killing I  - Phages</span></span></a></li>
			<li><a href="https://2008.igem.org/Team:Heidelberg/Project/Killing_II"><span><span>Killing II - Colicin</span></span></a></li>
			<li><a href="https://2008.igem.org/Team:Heidelberg/Project/Visualization"><span><span>Visualization</span></span></a></li>
			</ul>
			<!--[if lte IE 6]></td></tr></table></a><![endif]-->
		</li>

		<li>
			<a href="https://2008.igem.org/Team:Heidelberg/Parts" style="color: white">Parts<!--[if gt IE 6]><!--></a><!--<![endif]-->
			<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
				<ul class="DropDownMenu" id="MB1-DDM2">
				<li><a href="https://2008.igem.org/Team:Heidelberg/Parts"><span><span>Submitted Parts</span></span></a></li>
				<li><a href="https://2008.igem.org/Team:Heidelberg/Parts/Characterization"><span><span>Characterization</span></span></a></li>
				</ul>
				<!--[if lte IE 6]></td></tr></table></a><![endif]-->
		</li>
		<li>
			<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]-->
				<ul class="DropDownMenu" id="MB1-DDM3">
				<li><a href="https://2008.igem.org/Team:Heidelberg/Modeling"><span><span>Overview</span></span></a></li>
				<li><a href="https://2008.igem.org/Team:Heidelberg/Modeling/Chemotaxis"><span><span>Chemotaxis-Colicin</span></span></a></li>
				<li><a href="https://2008.igem.org/Team:Heidelberg/Modeling/Phage"><span><span>Phage Dynamics model</span></span></a></li>
				</ul>
				<!--[if lte IE 6]></td></tr></table></a><![endif]-->
		</li>
		<li>
			<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]-->
				<ul class="DropDownMenu" id="MB1-DDM5">
				<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook"><span><span>Sensing ></span></span><!--[if gt IE 6]><!--></a><!--<![endif]-->
			<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
					<ul class="SideMenu" id="MB1-DDM2-SM1">
					<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Cloning"><span>Cloning strategy</span></a></li>
					<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook"><span>Notebook</span></a></li>
					</ul>
					<!--[if lte IE 6]></td></tr></table></a><![endif]-->
				</li>
				
				<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Notebook"><span><span>Killing I - Phages ></span></span><!--[if gt IE 6]><!--></a><!--<![endif]-->
			<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
					<ul class="SideMenu" id="MB1-DDM2-SM2">
					<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Cloning"><span>Cloning strategy</span></a></li>
					<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Notebook"><span>Notebook</span></a></li>
					</ul>
					<!--[if lte IE 6]></td></tr></table></a><![endif]-->
				</li>
				<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Notebook"><span><span>Killing II - Colicin ></span></span><!--[if gt IE 6]><!--></a><!--<![endif]-->
			<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
					<ul class="SideMenu" id="MB1-DDM2-SM3">
					<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Cloning"><span>Cloning strategy</span></a></li>
					<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Notebook"><span>Notebook</span></a></li>
					</ul>
					<!--[if lte IE 6]></td></tr></table></a><![endif]-->
				</li>
				<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/visualization"><span><span>Visualization</span></span></a>
				<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/material"><span><span>Material & Methods</span></span></a>
				<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/team_meetings"><span><span>Team Meetings</span></span></a>
				<li><a href="https://2008.igem.org/Team:Heidelberg/Notebook/seminar"><span><span>Seminar on Synthetic Biology</span></span></a>
				</ul>
				<!--[if lte IE 6]></td></tr></table></a><![endif]-->
		</li>
		<li style="width: 160px">
			<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]-->
				<ul class="DropDownMenu" id="MB1-DDM4">
                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview"><span><span>Project Overview</span></span></a></li>
			       <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Phips_the_Phage"><span><span>Phips the Phage</span></span></a></li>
                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Essay"><span><span>Essay</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/Nobel_Prize"><span><span>Nobel Prize</span></span></a></li>				
				
				</ul>
				<!--[if lte IE 6]></td></tr></table></a><![endif]-->
		</li>		

		<li>
			<a href="https://2008.igem.org/Team:Heidelberg/Sponsors" style="color: white">Sponsors</a>
		</li>
	</ul>

</div>
<div id="header_bottom"><img src="https://static.igem.org/mediawiki/2008/e/ef/Navi_bg.gif" alt="" / ></div>
</body>
</html>

'''10<sup>th</sup> week'''

[[Team:Heidelberg/Notebook/Killing_II/9thweek | go back to 9<sup>th</sup> week]]

[[Team:Heidelberg/Notebook/Killing_II/Notebook |go back to the overview]]

==Monday 10/06/2008==
===pSB1A2-Receiver-Colicin cloning===

*Gel of  E9 lys PCR-Screen: 1% Agarose, 135 V, 35 min [[Image:081006-PCR_Screen_ColE9lys_standardization_small.jpg |500 px | center]]
*Gel-Results of E9-PCR-Screen: 
**Estimated Bands: ~2150bp
**Estimated Bands visible in many tubes -> Screening of single colonies.
*PCR-Screen of single colonies of pSB1A2-Receiver-Col E9 lys

 25.0 µl Taq Master Mix (Fermentas)
  2.5 µl T9002_LuxpR_Not_Eco_Xba_G_fw (T<sub>m</sub>=80,96°C)
  2.5 µl ColE9_lyProt_rv_A_SpeI (T<sub>m</sub>=64,29°C)
 20.0 µl H<sub>2</sub>O
    1    colony
 -------
 50.0 µl

 program:<br>
 95 °C    3 min
 95 °C    1 min          |
 64 °C    1 min          | 30 cycles
 72 °C    1 min 30 sec   |
 72 °C    10 min
  4 °C   constant

*Gel of  E9 lys PCR-Screen: 1% Agarose, 135 V, 35 min [[Image:081006-PCR_single_screen_colE9lys_Standardization_small.jpg |500 px | center]]
*Gel-Results of E9-PCR-Screen: Colonies 1, 2, 3, 4, 7 & 8 positive; Inoculation of 5 ml LB-Amp media with this colonies for miniprep, 37 °C -> ON
====Colicin Activity Test====
*Plan for activity tests of Colicin-Receivers:
**Overnight measurement (Monday - Tuesday) in Tecan plate reader with reference promotor cells (BBa_I20260) in combination with pSB1A2-Rec-ColE9 lys lysed supernatant.
**Plate scheme (click for detailed image): [[Image: 08-10-05_-_Colicintest_Col_E9_lys.jpg | 800 px | center]]

[[https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/10thweek back]]

==Tuesday 10/07/2008==
===pSB1A2-Receiver-Colicin cloning===
*Colicin-Activity-Test: Cultures were not grown -> new inoculation
*Miniprep of pSB1A2-Receiver-ColE9lys (BioBrick Standard): colonies 1, 2, 3, 4, 7 & 8, eluted in 35 µl H<sub>2</sub>O
*Digestion of pSB1A2-Receiver-ColE9lys (BioBrick Standard): colonies 1, 2, 3, 4, 7 & 8
 colonies 1 & 8: <br>
  5.0 µl DNA (~65-80 ng/µl)
 25.5 µl H<sub>2</sub>O
  4.0 µl BSA 10x (NEB)
  4.0 µl NEBuffer 2 (NEB)
  0.5 µl SpeI (NEB)
  1.0 µl XbaI (NEB)
 -------
 40.0 µl

 colonies 2, 3 & 4: <br>
  2.0 µl DNA (~245-275 ng/µl)
 28.5 µl H<sub>2</sub>O
  4.0 µl BSA 10x (NEB)
  4.0 µl NEBuffer 2 (NEB)
  0.5 µl SpeI (NEB)
  1.0 µl XbaI (NEB)
 -------
 40.0 µl

 colony 7: <br>
  2.0 µl DNA (~125 ng/µl)
 28.5 µl H<sub>2</sub>O
  4.0 µl BSA 10x (NEB)
  4.0 µl NEBuffer 2 (NEB)
  0.5 µl SpeI (NEB)
  1.0 µl XbaI (NEB)
 -------
 40.0 µl
*Gel of Digestion: 1% Agarose, 135 V, 30 min [[Image:081007-digestion_BB_Rec_colE9lys_small.jpg |500 px | center]]
*Digestion results:
**Expected fragments:
***2 x ~2000 (insert & vector), if XbaI site is recovered
***1 x ~4000, if XbaI site is not recovered
**In each colony there is only one fragment. Probably we have no positive clones with the BioBrick standard. To check the sequence we send the probes for sequencing.
*Send probes 1, 2, 3, 4, 7, & 8 to GATC for sequencing: Primers VF2 & VR
*Growth test of each Rec-Colicin glycerol stock: Inoculation in 2 ml LB-Amp and 2 ml TB-Amp. 37 °C -> ON






===Sender cloning: constitutive promotor-sender===
====Sender-activity-test====
**Inoculation of 15 ml TB-Amp media with sender (J23107 + F1610) from glycerol stock
**Inoculation of 15 ml TB-Amp media with GFP-receiver ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002])
**Inoculation of 15 ml LB-Amp media with sender (J23107 + F1610) from glycerol stock
**Inoculation of 15 ml LB-Amp media with GFP-receiver ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002])

*Plan for Wednesdaynesday:
**Inoculation of 7 ml TB media with 150 µl from sender ONC
**Inoculation of 7 ml TB media with 7 ml from GFP-receiver ONC
**Count amount of cells from ONC dilution t = 0 h (counting chamber)
**Measurement of optical density (OD) of ONC dilution t = 0 h
**Measurement of OD of one probe half-hourly for 8 - 10 h
**Creation of supernatant the measured probe by centrifugation and filtration half-hourly for 8 - 10 h
**Storing supernatant at 4 °C
**Count amount of cells from last probe (counting chamber)
**Start overnight measurement in Tecan plate reader
**plate scheme (click for detailed image): [[Image: 081007-pipetting_scheme_sender_test.jpg | 800 px | center ]]

[[https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/10thweek back]]

==Wednesday 10/08/2008==
===pSB1A2-Receiver-Colicin cloning===
====Standardization of pSB1A2-colE9lys-Receiver====
*Sequencing results: Colonies 1, 2, 3, 4 & 8 have the right sequence. Our first colicin Receiver fullfills BioBrick standard and is ready for activity tests. Colony 7 has mistakes inside the receiver and is thrown away.
*Digestion of pSB1A2-Receiver-ColE9lys colony 2 with XbaI/SpeI, XbaI, SpeI, EcoRI/PstI:
 'XbaI/SpeI:'
  5.0 µl DNA (~240 ng/µl)
  5.0 µl NEBuffer 2 (NEB)
  5.0 µl BSA 10x (NEB)
  0.5 µl XbaI (20 000 Units/ml, NEB)
  1.0 µl SpeI (10 000 Units/ml, NEB)
 33.5 µl H<sub>2</sub>O
 -------
 50.0 µl
 
 'XbaI:'
  5.0 µl DNA (~240 ng/µl)
  5.0 µl NEBuffer 2 (NEB)
  5.0 µl BSA 10x (NEB)
  0.5 µl XbaI (20 000 Units/ml, NEB)
 34.5 µl H<sub>2</sub>O
 -------
 50.0 µl

 'SpeI:'
  5.0 µl DNA (~240 ng/µl)
  5.0 µl NEBuffer 2 (NEB)
  5.0 µl BSA 10x (NEB)
  1.0 µl SpeI (10 000 Units/ml, NEB)
 34.0 µl H<sub>2</sub>O
 -------
 50.0 µl

 'EcoRI/PstI:'
  5.0 µl DNA (~240 ng/µl)
  5.0 µl NEBuffer 2 (NEB)
  5.0 µl BSA 10x (NEB)
  0.5 µl EcoRI (20 000 Units/ml, NEB)
  0.5 µl PstI (20 000 Units/ml, NEB)
 34.0 µl H<sub>2</sub>O
 -------
 50.0 µl



====Colicin Acitivity test====
*Inoculation of ONC for colicin activity test on Thursday, 2008-10-09:
**10 ml TB-Kana with reference promoter cells from glycerolstocks ([http://partsregistry.org/Part:BBa_I20260 BBa_I20260])
**10 ml TB-Amp with pSB1A2-Receiver, pSB1A2-Receiver-ColE9lys, pSB1A2-Receiver-ColE1 from Glycerolstocks



===Sender cloning: constitutive promotor-sender===
====Sender-activity-test====
**Inoculation of 7 ml TB media with 150 µl from sender ONC
**Inoculation of 7 ml TB media with 7 ml from GFP-receiver ONC
**Measurement of optical density (OD) of ONC dilution t = 0 h
**Measurement of OD of one probe half-hourly for 7 h
**Creation of supernatant the measured probe by centrifugation and filtration half-hourly for 8 - 10 h
**Storing supernatant at 4 °C
**Start overnight measurement in Tecan plate reader
**plate scheme (click for detailed image): [[Image: 081007-pipetting_scheme_sender_test.jpg | 800 px | center ]

[[https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/10thweek back]]

==Thursday 10/09/2008==
===pSB1A2-Receiver-Colicin cloning===
====Mutagenesis of pSB1A2-Receiver-Colicin E1/E9====
*Mutagenesis PCR:
 5.0  µl ''pfu'' buffer
 1.0  µl forward primer (10 µM)
 1.0  µl reverse primer (10 µM)
 1.0  µl dNTPs (12.5 mM, 50x)
 0.5  µl template DNA - 5 to 50 ng plasmid DNA
 40.5 µl milliQ water
 1.0  µl cloned ''pfu'' polymerase (Stratagene)

 95 °C   30 sec
 95 °C   30 sec    |
 55 °C   60 sec    | 16 cycles
 68 °C   11 min    |
  4 °C   constant

*Digestion with DpnI (NEB) to cut parental plasmid: Added 1 µl of DpnI to the finished Mutagenesis PCR. DpnI cuts at methylated GATC sites, so that only parental plasmids without the mutation are cutted. 2 h -> 37 °C
*PCR Purification Kit (Qiagen) to purify the plasmids.
*Transformation of prior mutagenesis: 5 µl per 100 µl ''E. coli'' TOP 10 competent cells
 - start thawing 100 µl competent ''E. coli'' TOP 10 cells on crushed ice
 - add 5 µl ligation
 - incubate on ice for 20 minutes
 - heat shock at 42 °C for 45 sec
 - incubate 2 min on ice
 - add 400 µl preheated LB media
 - incubate at 37 °C for 1 h, 500 rpm
 - Plate 200 µl on preheated LB-Amp plate
 - incubate overnight
*Controlgel of Mutagenesis: 1 % Agarose, 135 V, 30 min; DNA is visible -> probably the mutagenesis worked. <br> [[Image:081009-mutagenese_controll_gel_small.jpg |500 px | center]]




====Colicin Activity tests====
*10 am: Inocullation of TB media from ONC
**50 ml TB-Kana with 1.4 ml ONC of reference promoter cells ([http://partsregistry.org/Part:BBa_I20260 BBa_I20260])
**3 x 50 ml TB-Amp with 1.4 ml ONC of pSB1A2-Receiver, pSB1A2-Receiver-ColE9lys, pSB1A2-Receiver-ColE1
*12.30 am: OD -> 0.320. Separation of the 3 Receiver cultures into 8 tubes with 3 ml. Activation with 8 different Autoinducer([http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/K3007 detailed information, Sigma]) concentrations: 0 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM & 100 nM.
*8.00 pm: Preparing the test
**plate scheme: <br> [[Image:081009-plate_scheme_colicin_activity_test.jpg |500 px | center]]
**Preparation of probes: <br> [[Image:081009-plate_scheme_colicin_activity_test_mix.jpg |500 px | center]]
**10 pm start of measurement

===Sender cloning: constitutive promotor-sender===
====Sender-activity-test====
*Results of ON measurements: The graph shows the GFP intensities over time for the different supernatants and one AI-1 concentration (c = 1 nM). The GFP induction is much lower for the supernatants in comparison to the AI-1. Other tests with lower AI-1 concentration have to be performed to determine the AI-1 production rate. [[Image: 081008-sender_Activity_test_GFP_sup_besch_small.jpg | 700 px | center ]]

[[https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/10thweek back]]

==Friday 10/10/2008==
===pSB1A2-Receiver-Colicin cloning===
====Mutagenesis of pSB1A2-Receiver-Colicin E1/E9====
*Inoculation of liquid cultures from transformation of the first mutagenesis.
*Miniprep of liquidcultures: eluted in 35 µl H<sub>2</sub>O (Qiagen Miniprepkit)
*2nd mutagenesis PCR of Colicin E1 - Receiver (mutation of first PstI site):
*Mutagenesis PCR:
 5.0  µl ''pfu'' buffer
 1.0  µl forward primer (10 µM)
 1.0  µl reverse primer (10 µM)
 1.0  µl dNTPs (12.5 mM, 50x)
 0.5  µl template DNA - 5 to 50 ng plasmid DNA
 40.5 µl milliQ water
 1.0  µl Turbo ''pfu'' polymerase (Stratagene)

 95 °C   30 sec
 95 °C   30 sec    |
 55 °C   60 sec    | 16 cycles
 68 °C   11 min    |
  4 °C   constant

===Sender Cloning: constitutive promotor - sender===

[[https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/10thweek back]]

==Saturday 10/11/2008==

===Construction of reporterplasmid with kanamycin resistance===
====mCherry====
*For the visualization of our killer cells we need a RFP or mCherry reporter plasmid with kanamycin resistance. We decided to clone a cheY-mCherry fusion protein into pBAD18 vector.
*Digestion of pBAD18 plasmid & pES16-CheY-mCherry with SacI/XbaI: 2h 37 °C, 20 min 65 °C
 20.0 µl DNA
  0.5 µl SacI (20 000 U/ml, 100 % Activity in NEBuffer 4, NEB)
  0.7 µl XbaI (20 000 U/ml, 75 % Activity in NEBuffer 4, NEB)
  5.0 µl BSA 10x
  5.0 µl NEBuffer 4 (NEB)
 18.8 µl H<sub>2</sub>O
 -------
 50.0 µl
*Gel of Digestion: 0.7% Agarose, 135 V, 30 min [[Image:081011-cheY-mCherry_pBAD-digestion_small.jpg |500 px | center]]
*Gelresults:
**Expected Fragments:
***pBAD -> ~4600 bp
***CheY-mCherry -> ~1100 bp
**We were able to cut both fragments.
**Gelextraction with Qiagen Kit: eluted in 35 µl H<sub>2</sub>O
*Ligation of pBAD & mCherry: 11 h -> 16 °C, 20 min -> 65 °C
 upper band <br>
  2.0 µl T4 DNA Ligase Buffer (Fermentas)
  2.0 µl T4 DNA Ligase (Fermentas)
  0.5 µl pBAD (11.4 ng/µl) 
 15.5 µl CheY-mCherry (8.3 ng/µl) 
 -------
 20.0 µl
*EDIT 10/12/202008: calculated DNA ratio wrong


====RFP====
*In addition we try to clone RFP gene cassette from J23102 into pSB3K3.
**Digestion of BBa_J23102 & BBa_I15030 with EcoRI/PstI: 2 h -> 37 °C, 20 min -> 65 °C
 20.0 µl DNA
  1.0 µl EcoRI (20 000 U/ml, NEB)
  1.0 µl PstI (10 000 U/ml, NEB)
  5.0 µl BSA 10x
  5.0 µl EcoRI Buffer(NEB)
 18.5 µl H<sub>2</sub>O
 -------
 50.0 µl
*Gel of Digestion: 0.7% Agarose, 135 V, 30 min [[Image:081011-J23102___pSB3K3_small.jpg |500 px | center]]
*Gelresults:
**Expected Fragments:
***J23102 -> ~945 bp
***I15030 -> ~1909 bp
***pSB3K3 -> ~2750 bp
**J23102 was cutted out of the gel. For pSB3K3 we were not sure which was the right fragment so we go on with both fragments.
**Gelextraction with Qiagen Kit: eluted in 35 µl H<sub>2</sub>O
*Ligation of J23102 and pSB3K3 (5:1):
 upper band <br>
  2.0 µl T4 DNA Ligase Buffer (Fermentas)
  2.0 µl T4 DNA Ligase (Fermentas)
  8.5 µl pSB3K3 (upper Band) 
  7.5 µl BBa_J23102
 -------
 20.0 µl

 lower band <br>
  2.0 µl T4 DNA Ligase Buffer (Fermentas)
  2.0 µl T4 DNA Ligase (Fermentas)
 11.6 µl pSB3K3 (lower band) 
  4.4 µl BBa_J23102
 -------
 20.0 µl
*EDIT 10/12/202008: calculated DNA ratio wrong





===pSB1A2-Receiver-Colicin cloning===
====Mutagenesis of pSB1A2-Receiver-Colicin E1/E9====
*Digestion of overnight PCR probes with DpnI for 2 h -> 37 °C.
*PCR purification of probes: eluted in 35 µl H<sub>2</sub>O (Qiagen Kit)
*Transformation of prior mutagenesis: 5 µl per 100 µl ''E. coli'' TOP 10 competent cells
 - start thawing 100 µl competent ''E. coli'' TOP 10 cells on crushed ice
 - add 5 µl ligation
 - incubate on ice for 20 minutes
 - heat shock at 42 °C for 45 sec
 - incubate 2 min on ice
 - add 400 µl preheated LB media
 - incubate at 37 °C for 1 h, 500 rpm
 - Plate 200 µl on preheated LB-Amp plate
 - incubate overnight
*Controlgel of Mutagenesis: 1 % Agarose, 135 V, 30 min; DNA is visible -> probably the mutagenesis worked. <br> [[Image:081011-controlgel_2nd_mutagenesis.jpg |500 px | center]]
*Controlldigestion of first mutagenesis of Colicin E1 & E9 - Receiver with EcoRI: 2h -> 37 °C
 10.0 µl DNA
  2.0 µl BSA 10x (NEB)
  0.5 µl EcoRI (NEB)
  2.0 µl Buffer EcoRI 
  5.5 µl H<sub>2</sub>O
 -------
 20.0 µl
*Gel of Digestion: 1% Agarose, 135 V, 30 min [[Image:081011-controlgel_digestion_mutagenesis_small.jpg |500 px | center]]
*Gelresults: Mutated Colicin E1 and E9 Receivers were cutted only once by EcoRI. -> Mutagenesis sucessful.



====Colicin E9 operon - parts: Colicin E9====
*Digestion of Colicin E9 PCR product & pSB3K3 (from BBa_I15030)
 20.0 µl DNA
  1.0 µl EcoRI (20 000 U/ml, NEB)
  1.0 µl PstI (10 000 U/ml, NEB)
  5.0 µl BSA 10x
  5.0 µl EcoRI Buffer(NEB)
 18.5 µl H<sub>2</sub>O
 -------
 50.0 µl
*Gel of Digestion: 0.7% Agarose, 135 V, 30 min [[Image:081011-colicinE9-digestion_small.jpg |500 px | center]]
*Gelresults:
**Expected Fragments:
***Colicin E9 -> ~620 bp
**Colicin E9 was cutted out of the gel.
**Gelextraction with Qiagen Kit: eluted in 35 µl H<sub>2</sub>O
*Ligation of colicin E9 and pSB3K3 (5:1):
 upper band <br>
  2.0 µl T4 DNA Ligase Buffer (Fermentas)
  2.0 µl T4 DNA Ligase (Fermentas)
  6.1 µl pSB3K3 (upper Band) 
  9.9 µl Colicin E9
 -------
 20.0 µl

 lower band <br>
  2.0 µl T4 DNA Ligase Buffer (Fermentas)
  2.0 µl T4 DNA Ligase (Fermentas)
  9.5 µl pSB3K3 (lower band) 
  8.5 µl Colicin E9
 -------
 20.0 µl
EDIT 10/12/202008: calculated DNA ratio wrong









===Sender Cloning: constitutive promotor - sender===
====Sender activity test====
*Constitutive sender and amplifier ([http://partsregistry.org/Part:BBa_I15030 BBa_I15030]) activity test
**7x Inoculation of 7 ml TB media with 140 µl from sender ONC
**7x Inoculation of 7 ml TB media with 140 µl from amplifier ONC
**Inoculation of 7 ml TB media with 7 ml from GFP-receiver ONC
**Measurement of optical density (OD) of ONC dilutions t = 0 h
**Measurement of OD of one probe hourly for 6 h
**Creation of supernatant of the measured probe by centrifugation and filtration hourly for 6 h
**Storing supernatant at 4 °C
**Count amount of cells from last probe (counting chamber)
**plate scheme: <br> [[Image: 081011-plate_scheme_sender_amplifier_test.jpg | 800 px | center]]
**Unfortunately we were not able to measure ON because the Tecan plate reader was not recognized by the PC.

[[https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/10thweek back]]

==Sunday 10/12/2008==
===Construction of reporterplasmid with kanamycin resistance===
====mCherry====
*Transformation of pBAD-mCherry Ligation of saturday, 10/11/2008 (wrong vector-insert ratio): 5 µl per 100 µl ''E. coli'' TOP 10 competent cells
 - start thawing 100 µl competent ''E. coli'' TOP 10 cells on crushed ice
 - add 5 µl ligation
 - incubate on ice for 20 minutes
 - heat shock at 42 °C for 45 sec
 - incubate 2 min on ice
 - add 400 µl preheated LB media
 - incubate at 37 °C for 1 h, 500 rpm
 - Plate 500 µl on preheated LB-Amp plate
 - incubate overnight at 37°C
*Ligation of pBAD and mCherry (1:5): 1 h -> 22 °C, 20 min -> 65 °C
**Reason: Transformation on Saturdayurday was carried out with wrong ratio of vector and insert (calculation error).
  2.0 µl T4 DNA Ligase Buffer (Fermentas)
  2.0 µl T4 DNA Ligase (Fermentas)
 6.05 µl pBAD (11.4 ng/µl) 
 9.95 µl CheY-mCherry (8.3 ng/µl) 
 -------
 20.0 µl
*Transformation of pBAD-mCherry Ligation: 5 µl per 100 µl ''E. coli'' TOP 10 competent cells
 - start thawing 100 µl competent ''E. coli'' TOP 10 cells on crushed ice
 - add 5 µl ligation
 - incubate on ice for 20 minutes
 - heat shock at 42 °C for 45 sec
 - incubate 2 min on ice
 - add 400 µl preheated LB media
 - incubate at 37 °C for 1 h, 500 rpm
 - Plate 500 µl on preheated LB-Amp plate
 - incubate overnight at 37°C
====RFP====
*Transformation of J23102 and pSB3K3 Ligation of saturday, 10/11/2008 (wrong vector-insert ratio): 5 µl per 100 µl ''E. coli'' TOP 10 competent cells
 - start thawing 100 µl competent ''E. coli'' TOP 10 cells on crushed ice
 - add 5 µl ligation
 - incubate on ice for 20 minutes
 - heat shock at 42 °C for 45 sec
 - incubate 2 min on ice
 - add 400 µl preheated LB media
 - incubate at 37 °C for 1 h, 500 rpm
 - Plate 500 µl on preheated LB-Amp plate
 - incubate overnight at 37°C

===pSB1A2-Receiver-Colicin cloning===
====Mutagenesis of pSB1A2-Receiver-Colicin E1====
*Inoculation of Col E1 second mutagenesis step in 10 ml LB-amp; 5 h -> 37 °C
**Colonies 1-8 of Col E1 (5)
**Colonies 10-17 of Col E1 (1)
*Miniprep of liquidcultures (Col E1 (1.14;1.16;5.1;5.7): eluted in 35 µl H2O (Qiagen Miniprepkit)
*Controlldigestion of second mutagenesis of Colicin E1 - Receiver with PstI: 2 h -> 37 °C
 10.0 µl DNA (Miniprep)
  2.0 µl BSA 10x (NEB)
  0.5 µl PstI(NEB)
  2.0 µl Buffer EcoRI 
  5.5 µl H<sub>2</sub>O
 -------
 20.0 µl
*Gel of Digestion: 1% Agarose, 135 V, 30 min [[Image:081012-control_digestion_PstI_colE1_Rec_2nd_mut.jpg |500 px | center]]
*Gelresults: It can not be concluded weather the mutation worked. The fragments that are expected with and without the mutated PstI-Site are very similar.
*Third Mutagenesis PCR:
  5.0  µl ''pfu'' buffer
  1.0  µl forward primer (10 µM Col E1_mut_Pst_2_fw)
  1.0  µl reverse primer (10 µM Col E1_mut_Pst_2_rv)
  1.0  µl dNTPs (12.5 mM, 50x)
  0.5  µl template DNA - 5 to 50 ng plasmid DNA
 40.5 µl milliQ water
  1.0  µl turbo ''pfu'' polymerase (Stratagene)
  ----
  50.0 µl

 95 °C   30 sec
 95 °C   30 sec    |
 55 °C   60 sec    | 16 cycles
 68 °C   11 min    |
  4 °C   constant
*Digestion with DpnI (NEB) of the third mutagenesis to cut parental plasmid: Added 1 µl of DpnI to the finished Mutagenesis PCR. DpnI cuts at methylated GATC sites, so that only parental plasmids without the mutation are cutted. 2 h 35 min -> 37 °C
*Controlgel of third Mutagenesis (PstI_2): 1 % Agarose, 135 V, 30 min; DNA only visible in products from the colonie 1 (1.14 and 1.16) -> probably the mutagenesis worked on this plasmid. <br> [[Image:081012-3rd_mutagenesis_controll_gel_small.jpg |500 px | center]]
*PCR Purification Kit (Qiagen) to purify the plasmids. Eluated in 35 µl H<sub>2</sub>O 
*Transformation of prior mutagenesis: 5 µl per 100 µl ''E. coli'' TOP 10 competent cells
 - start thawing 100 µl competent ''E. coli'' TOP 10 cells on crushed ice
 - add 5 µl ligation
 - incubate on ice for 20 minutes
 - heat shock at 42 °C for 45 sec
 - incubate 2 min on ice
 - add 400 µl preheated LB media
 - incubate at 37 °C for 1 h, 500 rpm
 - Plate 500 µl on preheated LB-Amp plate
 - incubate overnight






====Standardization of pSB1A2-colE9plasmid-Receiver====
*PCR to add right BioBrick prefix and suffix to mutated ColicinE9-Receiver (without EcoRI site, colonies 2 & 4)
  2.5 µl Primer fw T9002 (10 µM)
  2.5 µl Primer rv (10 µM)
 18.0 µl H<sub>2</sub>O
 25.0 µl Phusion MasterMix (Finnzymes, NEB)
  2.0 µl Template (ColE9-Receiver mutated)
 -------
 50.0 µl

 98 °C    30 sec
 98 °C    10 sec          |
 56 °C    30 sec          | 25 cycles
 72 °C    1 min 30 sec    |
 72 °C    10 min
  4 °C    constant
*Gel to purify PCR products: 0.7%, 135 V, 30 min [[Image:081012-standardization_colE9_rec_gelex_PCR_small.jpg |500 px | center]]
*Gelextraction: eluted in 35 µl H<sub>2</sub>O (Qiagen, Gelextraction Kit)
*Digestion of ColE9-Receiver PCR product with EcoRI and SpeI: 2h -> 37 °C
  6.5 µl H<sub>2</sub>O
  5.0 µl BSA 10x (NEB)
  5.0 µl EcoRI Buffer (NEB)
  0.5 µl EcoRI (NEB)
  1.0 µl SpeI (NEB)
 32.0 µl DNA
 -------
 50.0 µl
*Ligation of ColicinE9-Receiver (BioBrick) with pSB1A2 (3:1): 1h -> 22 °C, 20 min 65 °C
 ColicinE9-Receiver (4)
  1.1 µl pSB1A2
 14.9 µl ColicinE9-Receiver
  2.0 µl T4 DNA Ligase
  2.0 µl T4 DNA Ligase Buffer
 -------
 20.0 µl

 ColicinE9-Receiver (2)
  1.8 µl pSB1A2
 14.2 µl ColicinE9-Receiver
  2.0 µl T4 DNA Ligase
  2.0 µl T4 DNA Ligase Buffer
 -------
 20.0 µl
*Transformation of prior ligation: 5 µl per 100 µl ''E. coli'' TOP 10 competent cells
 - start thawing 100 µl competent ''E. coli'' TOP 10 cells on crushed ice
 - add 5 µl ligation
 - incubate on ice for 20 minutes
 - heat shock at 42 °C for 45 sec
 - incubate 2 min on ice
 - add 400 µl preheated LB media
 - incubate at 37 °C for 1 h, 500 rpm
 - Plate 500 µl on preheated LB-Amp plate
 - incubate overnight

[[https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/10thweek back]]

[[Team:Heidelberg/Notebook/Killing_II/11thweek | go to 11<sup>th</sup> week]]