http://2009.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Muriel+aldunate&year=&month=2009.igem.org - User contributions [en]2024-03-28T15:23:21ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Victoria_Australia/SponsorsTeam:Victoria Australia/Sponsors2011-01-03T11:27:14Z<p>Muriel aldunate: /* We Would Like To Thank Our Generous Sponsors */</p>
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<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
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== We Would Like To Thank Our Generous Sponsors==<br />
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[[Image:RMIT.jpg|400px|thumb|RMIT University, Melbourne, Australia|left]] The Royal Melbourne Institute of Technology is one of Australia’s original and leading educational institutions with an international reputation for excellence in work-relevant education and high quality research.<br />
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[[Image:HIRi.gif|thumb|220px|RMIT Health Innovations Research Institute (HIRi), Melbourne|left]] RMIT HIRi takes an integrated approach to address key health issues facing the Australian community in the 21st century through research programs focusing on understanding how the human body functions at a molecular and cellular level. This knowledge will inform innovative therapeutic strategies to target disease states.<br />
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[[Image:csiro.jpg|thumb|200px|CSIRO|left]] The Commonwealth Scientific and Industrial Research Organisation is Australia's national science agency and one of the largest and most diverse research agencies in the world.<br />
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[[Image:SEH.gif|thumb|220px|RMIT College of Science, Engineering and Health, Melbourne|left]] The RMIT College of Science, Engineering and Health comprises of ten schools across two campuses offering programs that range from certificates through to PhDs.<br />
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[[Image:qiagen.gif|thumb|Qiagen|left]] QIAGEN is the leading provider of sample and assay technologies. Sample technologies are used to isolate DNA, RNA, and proteins from any biological sample. Assay technologies are then used to make specific target biomolecules, such as the DNA of a specific virus, visible for subsequent analysis.<br />
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'''ACKNOWLEDGMENTS:''' <br />
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We would also like to thank all individuals who contributed to the success of the RMIT University Fluoroforce team:<br />
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-Friends, family and teachers who provided moral support and guidance to the teams' Undergraduate students<br />
<br />
-Mr Kieran Wilson: Web Content Coordinator for the Science, Engineering & Health Office (RMIT University)<br />
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-Mr Robin Dick: Manager, Benefit Attraction. Alumni and Development (RMIT University)<br />
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-Bennetts Lane Jazz, Farm pro, Journal Cafe and Journal Canteen for their donations</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/SponsorsTeam:Victoria Australia/Sponsors2011-01-03T11:26:27Z<p>Muriel aldunate: /* We Would Like To Thank Our Generous Sponsors */</p>
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<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
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== We Would Like To Thank Our Generous Sponsors==<br />
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{| class="wikitable" border="0" cellpadding="10"<br />
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[[Image:RMIT.jpg|400px|thumb|RMIT University, Melbourne, Australia|left]] The Royal Melbourne Institute of Technology is one of Australia’s original and leading educational institutions with an international reputation for excellence in work-relevant education and high quality research.<br />
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[[Image:HIRi.gif|thumb|220px|RMIT Health Innovations Research Institute (HIRi), Melbourne|left]] RMIT HIRi takes an integrated approach to address key health issues facing the Australian community in the 21st century through research programs focusing on understanding how the human body functions at a molecular and cellular level. This knowledge will inform innovative therapeutic strategies to target disease states.<br />
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[[Image:csiro.jpg|thumb|200px|CSIRO|left]] The Commonwealth Scientific and Industrial Research Organisation is Australia's national science agency and one of the largest and most diverse research agencies in the world.<br />
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[[Image:SEH.gif|thumb|220px|RMIT College of Science, Engineering and Health, Melbourne|left]] The RMIT College of Science, Engineering and Health comprises of ten schools across two campuses offering programs that range from certificates through to PhDs.<br />
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[[Image:qiagen.gif|thumb|Qiagen|left]] QIAGEN is the leading provider of sample and assay technologies. Sample technologies are used to isolate DNA, RNA, and proteins from any biological sample. Assay technologies are then used to make specific target biomolecules, such as the DNA of a specific virus, visible for subsequent analysis.<br />
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'''ACKNOWLEDGMENTS:''' <br />
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We would also like to thank all individuals who contributed to the success of the RMIT University Fluoroforce team:<br />
<br />
-Friends, family and teachers who provided moral support and guidance to the teams' Undergradtuate students<br />
<br />
-Mr Kieran Wilson: Web Content Coordinator for the Science, Engineering & Health Office (RMIT University)<br />
<br />
-Mr Robin Dick: Manager, Benefit Attraction. Alumni and Development (RMIT University)<br />
<br />
-Bennetts Lane Jazz, Farm pro, Journal Cafe and Journal Canteen for their donations</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/SponsorsTeam:Victoria Australia/Sponsors2011-01-03T11:25:32Z<p>Muriel aldunate: /* We Would Like To Thank Our Generous Sponsors */</p>
<hr />
<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
<br />
== We Would Like To Thank Our Generous Sponsors==<br />
<br />
{| class="wikitable" border="0" cellpadding="10"<br />
|-<br />
[[Image:RMIT.jpg|400px|thumb|RMIT University, Melbourne, Australia|left]] The Royal Melbourne Institute of Technology is one of Australia’s original and leading educational institutions with an international reputation for excellence in work-relevant education and high quality research.<br />
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[[Image:HIRi.gif|thumb|220px|RMIT Health Innovations Research Institute (HIRi), Melbourne|left]] RMIT HIRi takes an integrated approach to address key health issues facing the Australian community in the 21st century through research programs focusing on understanding how the human body functions at a molecular and cellular level. This knowledge will inform innovative therapeutic strategies to target disease states.<br />
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<br><br />
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[[Image:csiro.jpg|thumb|200px|CSIRO|left]] The Commonwealth Scientific and Industrial Research Organisation is Australia's national science agency and one of the largest and most diverse research agencies in the world.<br />
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[[Image:SEH.gif|thumb|220px|RMIT College of Science, Engineering and Health, Melbourne|left]] The RMIT College of Science, Engineering and Health comprises of ten schools across two campuses offering programs that range from certificates through to PhDs.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br><br />
<br />
<br />
[[Image:qiagen.gif|thumb|Qiagen|left]] QIAGEN is the leading provider of sample and assay technologies. Sample technologies are used to isolate DNA, RNA, and proteins from any biological sample. Assay technologies are then used to make specific target biomolecules, such as the DNA of a specific virus, visible for subsequent analysis.<br />
<br />
<br />
<br />
<br />
<br />
'''ACKNOWLEDGMENTS:''' <br />
----<br />
<br />
We would also like to thank all individuals who contributed to the success of the RMIT University Fluoroforce team:<br />
<br />
-Friends, family and teachers who provided moral support and guidance to the teams' Undergradtuate students<br />
<br />
-Mr Kieran Wilson: Web Content Coordinator for the Science, Engineering & Health Office (RMIT University)<br />
<br />
-Mr Robin Dick: Manager, Benefit Attraction. Alumni and Development (RMIT University)<br />
<br />
-Bennetts Lane Jazz, Farm pro, Journal Cafe and Journal Canteen for their donations</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/CSSTeam:Victoria Australia/CSS2009-10-21T04:31:01Z<p>Muriel aldunate: </p>
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<div><html><br />
<style><br />
<br />
/*--------- CUSTOM STYLE -------------*/<br />
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#bodyContent {<br />
padding: 10px;<br />
}<br />
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body {<br />
background-color:#390142;<br />
background-image:url(https://static.igem.org/mediawiki/2009/3/34/Team_Victoria_wiki_background.jpg);<br />
background-repeat:repeat-x;<br />
}<br />
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/*<br />
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Script-Free 3-Level Menu 1.3.4<br />
www.CesarDaniel.info/Tech/ScriptFree3LevelMenu.html<br />
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Sections<br />
<br />
* Reset<br />
* Menu Bar<br />
* Drop-Down Menus<br />
* Side Menus<br />
* Browser Specific<br />
* Default Colors (Required)<br />
<br />
*/<br />
/*---------------------------------------------------------------------------------------- Reset */<br />
div.MenuBar,<br />
div.MenuBar ul,<br />
div.MenuBar li,<br />
div.MenuBar a,<br />
div.MenuBar table,<br />
div.MenuBar span {<br />
margin: 0;<br />
border: 0;<br />
padding: 0;<br />
z-index: 255;<br />
list-style-image: none;<br />
position: relative;<br />
}<br />
<br />
div.MenuBar ul ul li {<br />
position: relative;<br />
z-index: 300;<br />
}<br />
<br />
div.MenuBar ul li ul.DropDownMenu {<br />
display: none; /* Hides all drop-down menus. */<br />
}<br />
/*<br />
li:hover works in IE7 and FF2.<br />
a:hover works in IE6 and FF2.<br />
a:hover breaks li:hover in FF2.<br />
*/<br />
div.MenuBar ul li:hover ul.DropDownMenu li ul.SideMenu,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a ul.SideMenu {<br />
display: none; /* Hides all side menus. */<br />
}<br />
/*------------------------------------------------------------------------------------- Menu Bar */<br />
div.MenuBar {<br />
/* height: 25px; */ /* Total height 27px. */<br />
}<br />
div.MenuBar ul li {<br />
float: left;<br />
list-style-type: none !important;<br />
margin: 0 !important;<br />
position: relative; /* Sets the positioning context for each drop-down menu. */<br />
border: 1px solid #AAAAAA;<br />
}<br />
div.MenuBar ul li a {<br />
display: block;<br />
height: 17px; /* Keep height + padding-top + padding-bottom sync with the menu bar height. */<br />
padding-top: 4px;<br />
padding-bottom: 4px;<br />
padding-left: 1em; /* Sets the left space between top-level items. */<br />
padding-right: 1em; /* Sets the right space between top-level items. */<br />
text-decoration: none !important;<br />
}<br />
/*------------------------------------------------------------------------------ Drop-Down Menus */<br />
div.MenuBar ul li:hover ul.DropDownMenu,<br />
div.MenuBar ul li a:hover ul.DropDownMenu {<br />
display: block;<br />
width: 13em; /* Drop-down menu width.<br />
Use MenuTailor.css to customize. */<br />
padding: 1px; /* Sets the drop-down menu "effective border" width. */<br />
position: absolute;<br />
top: 25px; /* Places the drop-down menu under the menu bar.<br />
Keep it sync with the menu bar height. */<br />
left: 0; /* Aligns the drop-down menu to its top-level item. */<br />
}<br />
div.MenuBar ul li:hover ul.DropDownMenu li a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a {<br />
width: 13em; /* Keep it sync with the drop-down menu width.<br />
Use MenuTailor.css to customize. */<br />
padding-left: 0;<br />
padding-right: 0;<br />
z-index: 700;<br />
}<br />
ul.DropDownMenu li a span {<br />
display: block;<br />
padding-left: 5px; /* Sets the left space of each drop-down menu item. */<br />
padding-right: 0.25em; /* Sets the right space of each drop-down menu item. */<br />
text-align: right; /* Aligns the >> symbol to the right. */<br />
}<br />
ul.DropDownMenu li a span span {<br />
float: left; /* Aligns the text (back) to the left. */<br />
}<br />
/*----------------------------------------------------------------------------------- Side Menus */<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {<br />
display: block;<br />
width: 11em; /* Side menu width.<br />
Use MenuTailor.css to customize. */<br />
padding: 1px; /* Sets the side menu "effective border" width. */<br />
position: absolute;<br />
top: -1px; /* Aligns the side menu to its drop-down menu item.<br />
Keep it sync with the side menu "effective border" width. */<br />
left: 13em; /* Places the side menu to the right of the drop-down menu.<br />
Keep it sync with the drop-down menu width.<br />
Use MenuTailor.css to customize. */<br />
}<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a {<br />
width: 11em; /* Keep it sync with the side menu width.<br />
Use MenuTailor.css to customize. */<br />
}<br />
div.MenuBar ul li ul.DropDownMenu li ul.SideMenu li a span {<br />
padding-left: 1.5em; /* Sets the left space of each side menu item. */<br />
padding-right: 0.5em; /* Sets the right space of each side menu item. */<br />
text-align: left;<br />
}<br />
/*----------------------------------------------------------------------------- Browser Specific */<br />
* html div.MenuBar {<br />
position: relative; /* For IE55 and IE6.<br />
Hidden (* html) from non-IE browsers. */<br />
}<br />
* html div.MenuBar ul li a {<br />
float: left; /* For IE55 and IE6.<br />
Breaks O9.<br />
Hidden (* html) from non-IE browsers. */<br />
}<br />
* html ul.DropDownMenu li a:hover {<br />
cursor: hand; /* For IE55.<br />
Hidden (* html) from non-IE browsers. */<br />
}<br />
ul.DropDownMenu li a:hover {<br />
cursor: pointer; /* For IE6 and IE7.<br />
Hidding it (* html) from non-IE browsers breaks IE7! */<br />
}<br />
* html div.MenuBar a:hover {<br />
text-decoration: none; /* For IE55 and IE6.<br />
Hidden (* html) from non-IE browsers. */<br />
}<br />
* html div.MenuBar ul li table,<br />
* html div.MenuBar ul li table td {<br />
border: 0; /* For IE55 and IE6.<br />
Hidden (* html) from non-IE browsers. */<br />
}<br />
/*------------------------------------------------------------------------------- Default Colors */<br />
div.MenuBar {<br />
background-color: Menu;<br />
border-color: ButtonShadow;<br />
}<br />
div.MenuBar a {<br />
background-color: Menu; /* Top-level unselected items. */<br />
color: MenuText;<br />
}<br />
div.MenuBar ul li:hover a,<br />
div.MenuBar ul li a:hover {<br />
background-color: Highlight; /* Top-level selected item. */<br />
color: HighlightText;<br />
}<br />
/*...............................................................................................*/<br />
div.MenuBar ul li:hover ul.DropDownMenu,<br />
div.MenuBar ul li a:hover ul.DropDownMenu {<br />
background-color: ButtonShadow; /* Sets the drop-down menu "effective border" color. */<br />
}<br />
div.MenuBar ul li:hover ul.DropDownMenu li a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a {<br />
background-color: Menu; /* Drop-down menu unselected items.<br />
Sets the drop-down menu "effective background" color. */<br />
color: MenuText;<br />
}<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover {<br />
background-color: Highlight; /* Drop-down menu selected item. */<br />
color: HighlightText;<br />
}<br />
/*...............................................................................................*/<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {<br />
background-color: ButtonShadow; /* Sets the side menu "effective border" color. */<br />
}<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a {<br />
background-color: Menu; /* Side menu unselected items.<br />
Sets the side menu "effective background" color. */<br />
color: MenuText;<br />
}<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a:hover,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a:hover {<br />
background-color: Highlight; /* Side menu selected item. */<br />
color: HighlightText;<br />
}<br />
/*-----------------------------------------------------------------------------------------------*/<br />
<br />
table.calendar { margin: 0; padding: 10px; }<br />
table.calendar td { margin: 0; padding: 2px; vertical-align: top; }<br />
table.month .heading td { padding:2px; background-color:#d49fe1; color:#3C1859; text-align:center; font-size:120%; font-weight:bold; }<br />
table.month .dow td { color:#3C1859; text-align:center; font-size:110%; }<br />
table.month td.today { background-color:none; }<br />
table.month td {<br />
border: none;<br />
margin: 0;<br />
padding: 1pt 1.5pt;<br />
font-weight: bold;<br />
font-size: 8pt;<br />
text-align: right;<br />
background-color: #eee;<br />
}<br />
#bodyContent table.month a { background:none; padding:0 }<br />
.day-active { color:#0000cc }<br />
.day-empty { color:#cc0000 }<br />
<br />
<br />
</style><br />
</html></div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/PassportTeam:Victoria Australia/Passport2009-10-21T03:38:19Z<p>Muriel aldunate: </p>
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<div>[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
{{:Team:Victoria_Australia/CSS}}<br />
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[[Image:passport_australian.jpg]]<br />
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'''Welcome to our Passport page'''<br />
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<br />
This page is the entry portal that will enable you to track the travels and adventures of the RMIT University Fluoroforce team members. Originating from RMIT in Melbourne, Australia; a second home to most of the team members all the way to the Spring workshops held in Japan and before finally coming to an end at the iGEM Jamboree, MIT Boston, USA. Welcome aboard...</div>Muriel aldunatehttp://2009.igem.org/File:Muriel_igem_article2.jpgFile:Muriel igem article2.jpg2009-10-21T00:16:28Z<p>Muriel aldunate: </p>
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<div></div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Media_and_PublicationsTeam:Victoria Australia/Media and Publications2009-10-21T00:15:57Z<p>Muriel aldunate: </p>
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<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
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<div></div>Muriel aldunatehttp://2009.igem.org/File:Muriel_article2.jpgFile:Muriel article2.jpg2009-10-21T00:12:19Z<p>Muriel aldunate: uploaded a new version of "Image:Muriel article2.jpg": Reverted to version as of 23:08, 20 October 2009</p>
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<div></div>Muriel aldunatehttp://2009.igem.org/File:Muriel_article2.jpgFile:Muriel article2.jpg2009-10-21T00:10:03Z<p>Muriel aldunate: uploaded a new version of "Image:Muriel article2.jpg": Reverted to version as of 23:14, 20 October 2009</p>
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<div></div>Muriel aldunatehttp://2009.igem.org/File:Muriel_article2.jpgFile:Muriel article2.jpg2009-10-21T00:08:56Z<p>Muriel aldunate: uploaded a new version of "Image:Muriel article2.jpg": Reverted to version as of 23:27, 20 October 2009</p>
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<div></div>Muriel aldunatehttp://2009.igem.org/File:Muriel_article2.jpgFile:Muriel article2.jpg2009-10-20T23:29:44Z<p>Muriel aldunate: uploaded a new version of "Image:Muriel article2.jpg": Reverted to version as of 22:56, 20 October 2009</p>
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<div></div>Muriel aldunatehttp://2009.igem.org/File:Muriel_article2.jpgFile:Muriel article2.jpg2009-10-20T23:27:47Z<p>Muriel aldunate: uploaded a new version of "Image:Muriel article2.jpg"</p>
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<div></div>Muriel aldunatehttp://2009.igem.org/File:Muriel_article2.jpgFile:Muriel article2.jpg2009-10-20T23:15:34Z<p>Muriel aldunate: uploaded a new version of "Image:Muriel article2.jpg": Reverted to version as of 22:56, 20 October 2009</p>
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<div></div>Muriel aldunatehttp://2009.igem.org/File:Muriel_article2.jpgFile:Muriel article2.jpg2009-10-20T23:14:31Z<p>Muriel aldunate: uploaded a new version of "Image:Muriel article2.jpg"</p>
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<div></div>Muriel aldunatehttp://2009.igem.org/File:Muriel_article2.jpgFile:Muriel article2.jpg2009-10-20T23:08:55Z<p>Muriel aldunate: uploaded a new version of "Image:Muriel article2.jpg"</p>
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<div></div>Muriel aldunatehttp://2009.igem.org/File:Muriel_article2.jpgFile:Muriel article2.jpg2009-10-20T22:56:02Z<p>Muriel aldunate: </p>
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<div></div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Media_and_PublicationsTeam:Victoria Australia/Media and Publications2009-10-20T22:55:23Z<p>Muriel aldunate: </p>
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<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
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[[Image:fireflies_in_bedroom.jpg|center|frame|]]<br />
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[[Image:muriel_article2.jpg|center|frame|]]</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Passport/Spring_WorkshopTeam:Victoria Australia/Passport/Spring Workshop2009-10-20T16:35:05Z<p>Muriel aldunate: </p>
<hr />
<div>[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
{{:Team:Victoria_Australia/CSS}}<br />
<br />
[[Image:Japan_header.jpg|center]]<br />
<br />
[[Image:tokyo_metro.jpg|200px|thumb|Challenge one - navigating the Tokyo Metro|right]]<br />
[[Image:IMGP2292.jpg|200px|thumb|Challenge two- navigating the University of Tokyo campus map!|right]]<br />
[[Image:tokyo_event.jpg|200px|thumb|Spring workshop itinerary|right]]<br />
[[Image:IMGP2295.jpg|200px|thumb|Three wise men of iGEM|right]]<br />
<br />
----<br />
<br />
<br />
<br />
'''The trip to Tokyo began with a mad dash for the two students attending, Muriel Aldunate and Candice Raeburn, as they rushed to the airport straight from their last exam. As iGEM began in USA and expanded to become an international competition, sometimes the dates and deadlines can be a little tricky for those not following the USA semester structure. Nonetheless the two students arrived at Narita thoroughly exhausted and excited to seek out their instructor, Dr. Leonard Pattenden, who had flown out the day before. After a couple of “detours” including purchasing the wrong tickets, cockroaches in inconvenient places, nearly being run over and with the help of a friendly convenience store clerk who left work to play navigator, the girls eventually found their way to their accommodation, forgot to take off their shoes, and slumped into bed to slumber the journey away in preparation for the iGEM 2009 Spring workshop.'''<br />
<br />
<br />
'''The next morning saw more disorientation as the group tried to navigate its way around the Tokyo. After walking past their destination and trying to make sense of a plethora of maps, a kind stranger pointed them in the right direction and they finally made it to the Takeda Building – or did they? In the knowledge they would probably get lost (and perhaps among mis-setting of watches) the group left so early they had arrived even before the iGEM organisers, wandering around the grounds until eventually some other lost-looking souls appeared with whom they could coalesce and talk about all things iGEM.'''<br />
<br />
<br />
'''"Synthetic biology is nanotecnology that works" Tom Knight begins by speaking passionately about the field that stole him away from what could have been a life of straight computer science. Tom and his team from MIT went on to give a little history and background on the roots of Synthetic Biology, the evolution of iGEM and the BioBrick concept. After a quick lunch it was back to the drawing board as we went through the navigation of the competition itself – forming ideas, setting achievable goals, using the iGEM website interface and construction using the BioBricks provided. We were then led by our kind hosts at Tokyo University through the awe-inspiring Ueno park to a restaurant where the teams could mingle over food and sake.'''<br />
<br />
<br />
'''Day two started with the breakfast of champions - sticky rice balls wraped in seaweed filled with seasoned meat from 7/11 and hot coffee from a vending machine (where you can also purchase liquor if you insert a valid ID). The workshop (much easier to find this time) continued with detailed instructions on BioBricks and the procedure for submitting to the registry. Thoughout the workshop, participants are called upon to be a part of “team example” where they can go through the steps in real time – a brilliant idea which made the process both understandable and memorable. A wonderful lunch of Bento boxes was provided, beverages among which were “Calpis” (pronounced cow-piss which met many giggles from the Australian teams) and ice cold tea. Finally safety was addressed, iGEM partners and special deals were announced, awards were detailed and the Asia workshop was completed.'''<br />
<br />
<br />
'''The RMIT and UQ teams then went shopping for robots in Akihabara then parted ways, filled with notes on their rivals and a few whisps of comradery too. Dr Pattenden boarded a train back to Narita to return home, and Candi and Muriel accidently caught the wrong train to Nikko to begin their Japanese adventure.'''<br />
<br />
<br />
[[Image:tri_pannel.jpg|left]]<br />
[[Image:muriel_kyoto_uni.jpg|200px|thumb|Dining with members of the Kyoto University iGEM team|left]][[Image:muriel_friends_kyoto_uni.jpg|200px|thumb|Making friends with members of the Kyoto University iGEM team|left]]</div>Muriel aldunatehttp://2009.igem.org/File:Muriel_kyoto_uni.jpgFile:Muriel kyoto uni.jpg2009-10-20T16:30:35Z<p>Muriel aldunate: </p>
<hr />
<div></div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Passport/Spring_WorkshopTeam:Victoria Australia/Passport/Spring Workshop2009-10-20T16:29:21Z<p>Muriel aldunate: </p>
<hr />
<div>[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
{{:Team:Victoria_Australia/CSS}}<br />
<br />
[[Image:Japan_header.jpg|center]]<br />
<br />
[[Image:tokyo_metro.jpg|200px|thumb|Challenge one - navigating the Tokyo Metro|right]]<br />
[[Image:IMGP2292.jpg|200px|thumb|Challenge two- navigating the University of Tokyo campus map!|right]]<br />
[[Image:tokyo_event.jpg|200px|thumb|Spring workshop itinerary|right]]<br />
[[Image:IMGP2295.jpg|200px|thumb|Three wise men of iGEM|right]]<br />
<br />
----<br />
<br />
<br />
<br />
'''The trip to Tokyo began with a mad dash for the two students attending, Muriel Aldunate and Candice Raeburn, as they rushed to the airport straight from their last exam. As iGEM began in USA and expanded to become an international competition, sometimes the dates and deadlines can be a little tricky for those not following the USA semester structure. Nonetheless the two students arrived at Narita thoroughly exhausted and excited to seek out their instructor, Dr. Leonard Pattenden, who had flown out the day before. After a couple of “detours” including purchasing the wrong tickets, cockroaches in inconvenient places, nearly being run over and with the help of a friendly convenience store clerk who left work to play navigator, the girls eventually found their way to their accommodation, forgot to take off their shoes, and slumped into bed to slumber the journey away in preparation for the iGEM 2009 Spring workshop.'''<br />
<br />
<br />
'''The next morning saw more disorientation as the group tried to navigate its way around the Tokyo. After walking past their destination and trying to make sense of a plethora of maps, a kind stranger pointed them in the right direction and they finally made it to the Takeda Building – or did they? In the knowledge they would probably get lost (and perhaps among mis-setting of watches) the group left so early they had arrived even before the iGEM organisers, wandering around the grounds until eventually some other lost-looking souls appeared with whom they could coalesce and talk about all things iGEM.'''<br />
<br />
<br />
'''"Synthetic biology is nanotecnology that works" Tom Knight begins by speaking passionately about the field that stole him away from what could have been a life of straight computer science. Tom and his team from MIT went on to give a little history and background on the roots of Synthetic Biology, the evolution of iGEM and the BioBrick concept. After a quick lunch it was back to the drawing board as we went through the navigation of the competition itself – forming ideas, setting achievable goals, using the iGEM website interface and construction using the BioBricks provided. We were then led by our kind hosts at Tokyo University through the awe-inspiring Ueno park to a restaurant where the teams could mingle over food and sake.'''<br />
<br />
<br />
'''Day two started with the breakfast of champions - sticky rice balls wraped in seaweed filled with seasoned meat from 7/11 and hot coffee from a vending machine (where you can also purchase liquor if you insert a valid ID). The workshop (much easier to find this time) continued with detailed instructions on BioBricks and the procedure for submitting to the registry. Thoughout the workshop, participants are called upon to be a part of “team example” where they can go through the steps in real time – a brilliant idea which made the process both understandable and memorable. A wonderful lunch of Bento boxes was provided, beverages among which were “Calpis” (pronounced cow-piss which met many giggles from the Australian teams) and ice cold tea. Finally safety was addressed, iGEM partners and special deals were announced, awards were detailed and the Asia workshop was completed.'''<br />
<br />
<br />
'''The RMIT and UQ teams then went shopping for robots in Akihabara then parted ways, filled with notes on their rivals and a few whisps of comradery too. Dr Pattenden boarded a train back to Narita to return home, and Candi and Muriel accidently caught the wrong train to Nikko to begin their Japanese adventure.'''<br />
<br />
<br />
[[Image:tri_pannel.jpg|left]]<br />
[[Image:muriel_kyoto_uni.jpg|200px|thumb|Dining with members of the Kyoto University iGEM team|left]]<br />
[[Image:muriel_friends_kyoto_uni.jpg|200px|thumb|Making friends with members of the Kyoto University iGEM team|left]]</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Passport/Spring_WorkshopTeam:Victoria Australia/Passport/Spring Workshop2009-10-20T16:17:50Z<p>Muriel aldunate: </p>
<hr />
<div>[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
{{:Team:Victoria_Australia/CSS}}<br />
<br />
[[Image:Japan_header.jpg|center]]<br />
<br />
[[Image:tokyo_metro.jpg|200px|thumb|Challenge one - navigating the Tokyo Metro|right]]<br />
[[Image:IMGP2292.jpg|200px|thumb|Challenge two- navigating the University of Tokyo campus map!|right]]<br />
[[Image:tokyo_event.jpg|200px|thumb|Spring workshop itinerary|right]]<br />
[[Image:IMGP2295.jpg|200px|thumb|Three wise men of iGEM|right]]<br />
<br />
<br />
<br />
'''The trip to Tokyo began with a mad dash for the two students attending, Muriel Aldunate and Candice Raeburn, as they rushed to the airport straight from their last exam. As iGEM began in USA and expanded to become an international competition, sometimes the dates and deadlines can be a little tricky for those not following the USA semester structure. Nonetheless the two students arrived at Narita thoroughly exhausted and excited to seek out their instructor, Dr. Leonard Pattenden, who had flown out the day before. After a couple of “detours” including purchasing the wrong tickets, cockroaches in inconvenient places, nearly being run over and with the help of a friendly convenience store clerk who left work to play navigator, the girls eventually found their way to their accommodation, forgot to take off their shoes, and slumped into bed to slumber the journey away in preparation for the iGEM 2009 Spring workshop.'''<br />
<br />
<br />
'''The next morning saw more disorientation as the group tried to navigate its way around the Tokyo. After walking past their destination and trying to make sense of a plethora of maps, a kind stranger pointed them in the right direction and they finally made it to the Takeda Building – or did they? In the knowledge they would probably get lost (and perhaps among mis-setting of watches) the group left so early they had arrived even before the iGEM organisers, wandering around the grounds until eventually some other lost-looking souls appeared with whom they could coalesce and talk about all things iGEM.'''<br />
<br />
<br />
'''"Synthetic biology is nanotecnology that works" Tom Knight begins by speaking passionately about the field that stole him away from what could have been a life of straight computer science. Tom and his team from MIT went on to give a little history and background on the roots of Synthetic Biology, the evolution of iGEM and the BioBrick concept. After a quick lunch it was back to the drawing board as we went through the navigation of the competition itself – forming ideas, setting achievable goals, using the iGEM website interface and construction using the BioBricks provided. We were then led by our kind hosts at Tokyo University through the awe-inspiring Ueno park to a restaurant where the teams could mingle over food and sake.'''<br />
<br />
<br />
'''Day two started with the breakfast of champions - sticky rice balls wraped in seaweed filled with seasoned meat from 7/11 and hot coffee from a vending machine (where you can also purchase liquor if you insert a valid ID). The workshop (much easier to find this time) continued with detailed instructions on BioBricks and the procedure for submitting to the registry. Thoughout the workshop, participants are called upon to be a part of “team example” where they can go through the steps in real time – a brilliant idea which made the process both understandable and memorable. A wonderful lunch of Bento boxes was provided, beverages among which were “Calpis” (pronounced cow-piss which met many giggles from the Australian teams) and ice cold tea. Finally safety was addressed, iGEM partners and special deals were announced, awards were detailed and the Asia workshop was completed.'''<br />
<br />
<br />
'''The RMIT and UQ teams then went shopping for robots in Akihabara then parted ways, filled with notes on their rivals and a few whisps of comradery too. Dr Pattenden boarded a train back to Narita to return home, and Candi and Muriel accidently caught the wrong train to Nikko to begin their Japanese adventure.'''<br />
<br />
<br />
[[Image:tri_pannel.jpg|left]]<br />
[[Image:muriel_friends_kyoto_uni.jpg|200px|thumb|Making friends with members of the Kyoto University iGEM team|left]]</div>Muriel aldunatehttp://2009.igem.org/File:Muriel_friends_kyoto_uni.jpgFile:Muriel friends kyoto uni.jpg2009-10-20T16:14:26Z<p>Muriel aldunate: </p>
<hr />
<div></div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Passport/Spring_WorkshopTeam:Victoria Australia/Passport/Spring Workshop2009-10-20T16:09:22Z<p>Muriel aldunate: </p>
<hr />
<div>[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
{{:Team:Victoria_Australia/CSS}}<br />
<br />
[[Image:Japan_header.jpg|center]]<br />
<br />
[[Image:tokyo_metro.jpg|200px|thumb|Challenge one - navigating the Tokyo Metro|right]]<br />
[[Image:IMGP2292.jpg|200px|thumb|Challenge two- navigating the University of Tokyo campus map!|right]]<br />
[[Image:tokyo_event.jpg|200px|thumb|Spring workshop itinerary|right]]<br />
[[Image:IMGP2295.jpg|200px|thumb|Three wise men of iGEM|right]]<br />
<br />
<br />
<br />
'''The trip to Tokyo began with a mad dash for the two students attending, Muriel Aldunate and Candice Raeburn, as they rushed to the airport straight from their last exam. As iGEM began in USA and expanded to become an international competition, sometimes the dates and deadlines can be a little tricky for those not following the USA semester structure. Nonetheless the two students arrived at Narita thoroughly exhausted and excited to seek out their instructor, Dr. Leonard Pattenden, who had flown out the day before. After a couple of “detours” including purchasing the wrong tickets, cockroaches in inconvenient places, nearly being run over and with the help of a friendly convenience store clerk who left work to play navigator, the girls eventually found their way to their accommodation, forgot to take off their shoes, and slumped into bed to slumber the journey away in preparation for the iGEM 2009 Spring workshop.'''<br />
<br />
<br />
'''The next morning saw more disorientation as the group tried to navigate its way around the Tokyo. After walking past their destination and trying to make sense of a plethora of maps, a kind stranger pointed them in the right direction and they finally made it to the Takeda Building – or did they? In the knowledge they would probably get lost (and perhaps among mis-setting of watches) the group left so early they had arrived even before the iGEM organisers, wandering around the grounds until eventually some other lost-looking souls appeared with whom they could coalesce and talk about all things iGEM.'''<br />
<br />
<br />
'''"Synthetic biology is nanotecnology that works" Tom Knight begins by speaking passionately about the field that stole him away from what could have been a life of straight computer science. Tom and his team from MIT went on to give a little history and background on the roots of Synthetic Biology, the evolution of iGEM and the BioBrick concept. After a quick lunch it was back to the drawing board as we went through the navigation of the competition itself – forming ideas, setting achievable goals, using the iGEM website interface and construction using the BioBricks provided. We were then led by our kind hosts at Tokyo University through the awe-inspiring Ueno park to a restaurant where the teams could mingle over food and sake.'''<br />
<br />
<br />
'''Day two started with the breakfast of champions - sticky rice balls wraped in seaweed filled with seasoned meat from 7/11 and hot coffee from a vending machine (where you can also purchase liquor if you insert a valid ID). The workshop (much easier to find this time) continued with detailed instructions on BioBricks and the procedure for submitting to the registry. Thoughout the workshop, participants are called upon to be a part of “team example” where they can go through the steps in real time – a brilliant idea which made the process both understandable and memorable. A wonderful lunch of Bento boxes was provided, beverages among which were “Calpis” (pronounced cow-piss which met many giggles from the Australian teams) and ice cold tea. Finally safety was addressed, iGEM partners and special deals were announced, awards were detailed and the Asia workshop was completed.'''<br />
<br />
<br />
'''The RMIT and UQ teams then went shopping for robots in Akihabara then parted ways, filled with notes on their rivals and a few whisps of comradery too. Dr Pattenden boarded a train back to Narita to return home, and Candi and Muriel accidently caught the wrong train to Nikko to begin their Japanese adventure.'''<br />
<br />
<br />
[[Image:tri_pannel.jpg|left]]</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Project/Materials_and_MethodsTeam:Victoria Australia/Project/Materials and Methods2009-10-20T15:33:32Z<p>Muriel aldunate: /* Mutagenesis of YFP */</p>
<hr />
<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
<br />
'''''<br />
== <div style="width: 60%; height: 60%; color: #50cad; font-size: 25px; ">Materials & Methods</div> ==<br />
'''''<br />
<br />
__TOC__<br />
==Transformation of fluorescent cells==<br />
{|style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top"<br />
|width="50%" |To begin with, all of the required reagent solutions were prepared. LB Agar plates (protocol 1) were made using 10g tryptone digest of casein, 5g yeast extract, 5g NaCl, 15g agar and then made up to 1L using milliQ water. Ampicillin (100μg/ml) was then prepared by adding 1gram of ampicillin and 9 ml (9gram) milliQ water so that the total volume was 10ml. LB media was made using 10g tryptone, 5g yeast extract, 5g NaCl and milliQ water to make the media up to 1L. <br><br />
<br />
The bacterial transformation of both BFP and GFP was completed by firstly Pre-incubating the LB-agar plates at 37°C for 2 hours prior. The Ca-competent cells were thawed on ice (4ºC). <br />
|width="50%"|Plasmid DNA was added and the tubes stored on ice for 30mins. The tubes were transferred to a rack placed in a preheated water bath (45ºC) and incubated for 30secs. The tubes were then transferred to ice (4ºC) and incubated on ice for 2 min. 800μl of SOC medium was added to each tube and Incubated at 37ºC with gentle shaking (no more that 50rpm) for 45min. 50μl of the transformed cells was transferred onto the LB agar plates each containing ampicillin, and spread the cell suspension thoroughly on the surface using a sterile rod (Streaking). Plates were incubated (agar side up) at 37ºC for ~16h. <br />
Transformation of Vic GFP, BFP, YFP, AZ, blueberry and cherry was done using the same protocol as above. <br />
<br><br />
|}<br />
<br />
==Protein expression==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|Following the transformation, protein expression was carried out by adding 100ml of Lb media into two conical flasks that were previously autoclaved. Using a pipette tip a single colony from the transformed bacteria of GFP and BFP was selected and dropped into conical flask. They were then incubated at 240rpm at 38 ºC for 18 hours.<br />
Using the incubated GFP and BFP they were divided into two falcon tubes each and centrifuged at 3000rpm for 20min. The supernatant was emptied (waste) and the pellet was resuspended with 20ml of SOC solution and 20 µL of ampicillin being added. At a wavelength of 600nm, the solution was measured in the spectrometer to check the density of the bacteria.The results obtained were as follows: GFP = 0.93A and BFP = 0.95A.Two conical flasks were made <br />
|width="50%" |up with 120ml SOC solution and 120 µL of Ampicillin (this is the solution in which GFP and BFP are added to).<br />
Using C1V1=C2V2 the amount of GFP and BFP that was added to the solution from above (SOC and Ampicillin) was found.After adding the GFP/BFP the solution was incubated at 240rpm at 28 ºC for 1 hour. Following this hour the absorbance was read at 600nm and the following results were obtained: GFP = 0.7A (undiluted) and BFP = 0.64A (undiluted).<br />
The proteins were then induced by adding 147µL of IPTG into each conical flask. This was poured into 2 50 ml falcon tubes and centrifuged for 10 min at 2000rpm. Stored at -80°C. <br />
<br />
|}<br />
<br />
==Preparation of reagents==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer. <br />
|width="50%" |100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution. 1M imieazole was prepared by adding 0.6808g of imieazole to 10ml of milliQ water. <br />
|}<br />
==Purification of GFP and BFP==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or BFP. That is; GFP with lysis buffer added to pellet with GFP and BFP with lysis buffer added to pellet with BFP. These were sonicated to split the cells open at 70% duty cycle 30seconds on and 20seconds off for 3 cycles. Samples then centrifuged for 40min. The supernatant was transferred into eppendorf tubes (2 times GFP and 2times BFP). 250 µL of resin was added to each tube and placed on suspension mixer for 20min. again, the supernatant was removed and added 1ml phosphate buffer to the remaining resin. <br />
This was centrifuged at 500rpm for 2min and the supernatant removed with 1ml phosphate buffer being again added. This was centrifuged at 500rpm for 2min and the supernatant removed. The pellet was resuspended with 5ml elution buffer (An elution buffer was made using 2.5ml imieazole with phosphate buffer being added resulting in a total volume of 10ml. ) again this was centrifuged for 5min at 500rpm. The resulting supernatant formed contained the GFP and BFP; this was removed. <br />
<br />
<br />
|width="50%"|<br />
[[Image:SNC00184.jpg|300px|centre|Purified GFP and BFP obtained in the supernatant|thumb]]<br />
|}<br />
<br />
==Preparation of yellow plasmid with E coli bacteria==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|A 6M guanidine hydrochloride solution was prepared along with 50ml Tris Buffer at pH 7.8 ( 1M Tris-HCL with the pH adjusted by adding NaOH). 4M Guanidinium in mix with buffer was added to the protein solution and spun for 30 minutes. This was then centrifuged at 20233rcf and the supernatant put in eppendorf tubes with resin. The ampicillin was then incubated with 1 micro litre/ml working stock 100µg/ml. <br />
4ml of LB medium was put into a flacon tube. With a pipette tip E.coli bacteria was scraped and placed into the falcon tube. It was then incubated overnight at 37 ºC 300rpm. 100ml 2YT solution added to 4 conical flasks. 1ml of the incubated Bactria stock (from above) was put into each flask and incubated at 180rpm 37 ºC. <br />
|width="50%" |After incubation it was centrifuged at 2880rpm for 20min and the supernatant poured into a conical flask (2YT) with decon disinfectant to kill off any remaining bacteria. 8 falcon tubes with the bacteria pellet were then stored (frozen). 6ml of LB media was added to 4 times 15ml falcon tubes. 0.1µl/nl = 6C ampicillin was put in and a stab of yellow fluorescent protein (3.1) was then added to the solution and resuspended. This was incubated at 190rpm at 37 ºC for 16 hours. Following incubation, the solution was centrifuged at 21000rpm for 5 min.<br />
|}<br />
<br />
==Purification of yellow fluorescent plasmid==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|The aurum plasmid spin format protocol was completed (The plasmid containing bacterial host of the yellow protein was transferred to a 1.5-2.0ml capped microcentrifuge tube. Cells were pelleted for 1 min at full speed and the supernatant removed. 250µL of resuspension solution was added and pipetted up and down until the cell pellet was completely resuspended. 250µL of lysis solution was added and mixed by inverting. 5 min after the lysis was added, 350µL of neutralisation solution was added and also mixed by inversion. The neutralised lysate was centrifuged for 5min at 21000rpm {the supernatant or cleared lysate contains the plasmid DNA}.<br />
While centrifuging, a plasmid mini column was inserted into a 2ml capless wash tube. The supernatant was transferred to the plasmid mini column and centrifuged for 1min.<br />
|width="50%" |Wash solution at 5X concentrate was used to add 4 volumes (100ml) of 95-100% ethanol. The plasmid mini column from the wash tube was removed and the filtrate discarded from the tube. The column was replaced into the same wash tube. 750µL of the wash solution was added and centrifuged for 1min. The wash solution was discarded from the tube and the column was replaced into the same wash tube. It was then centrifuged for an additional 1 min to remove the residual wash solution. The plasmid mini column was transferred to a 1.5-2.0ml capped microcentrifuge tube and <br />
50µL of elution solution was added onto the membrane stack at the base of the column and allowed a minute for the solution to saturate the membranes. It was again centrifuged for 1min to elute the plasmid. The mini column was discarded and the eluted DNA was stored in the freezer).<br />
|}<br />
<br />
==Preparation of the E.coli cell free system==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|The E.Coli cell free system was constructed via the following method:<br />
Two buffers s30a and s30b were first made (1.725g potassium acetate, 2.944g magnesium sulphate, added 400ml MQ water and set to a pH of 7.8 by adding trisacetate. It was then filtered in to 2X 250ml beakers one being for S30A in which 500µL of 2 mercaptoethanol was added and S30B that did not contain 2 mercaptoehanol).<br />
The bacteria cells were thawed on ice and resuspended the pellets by washing out with S30A buffer and all he solution transferred into 2X falcon tubes. <br />
|width="50%" |These were then centrifuged at 2000 RCF for 10 minutes. Again, the pellets were resuspended with S30A buffer and centrifuged at 2000 RCF for 10 minutes. This was completed twice as to a total of 3 times. The supernatant was removed. The pellet was resuspended with S30B buffer and then sonicated for 30 sec on and 30 sec off at 50% for a total of 5 minutes. <br />
1ml samples were placed 10 eppendorf tubes and centrifuged at 20 000 for 30 minutes at 4°C. Supernatant was transferred into clean eppendorf tubes and also centrifuged at 20000 rpm for 45 minutes. 7.25ml of the supernatant was removed to serve as the cell lysate in the E.coli cell free system. <br />
|}<br />
<br />
==Expression and purification of yellow and green fluorescent protein==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|An expression and purification of the yellow and green fluorescent proteins was carried out to determine what cell line it had; DH5 alpha or BL21DE3.<br />
An overnight culture was set up in 2X falcon tubed with 5ml LB media and a pipette that was stabbed with YFP and GFP. 5µL of ampicillin was added and incubated overnight at 37°C at 220rpm. The protein expression was then completed by dividing the incubated YFP and GFP into 2X falcon tubes and centrifuged for 20 minutes at 3000rpm. <br />
The supernatant was emptied and the pellet resuspended with 20 ml of SOC solution and 20µL of ampicillin added. <br />
Spectrophotemetric analysis of the GFP and YFP at 1:100 dilution was done in the nanodrop 2000. <br />
|width="50%" |Absorbance at 600nm was taken before the dilution and the results were as follows: GFP=1.4 and YFP=0.8. the overnight cultures were then spun in the centrifuge to create a pellet for resuspension and again incubated at 37°C and 220rpm. The OD measurement was taken to be: YFP = 0.195 and GFP = 0.374.<br />
The proteins were then induced by adding 147µL of IPTG into each conical flask containing the YFP and GFP. This was poured into 2 x 50 ml falcon tubes, centrifuged for 10 min at 2000rpm and subsequently stored at -80°C.<br />
|}<br />
<br />
==Electrophoresis of YFP==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|To measure the purity in preparation for the electrophoresis run of the YFP protein, the following reagents were prepared: 6 X DNA gel loading dye (5.6ml of 80% glycerol, 9.2ml MQ water, 0.18ml EDTA at 0.5M and 0.018g bromphenol blue; made up to a volume of 15ml) and agarose gel (0.5g DNA grade agarose, 50µL TAE buffer, 2µL cybersafe to 20µL MQ water and 5µL of agarose gel). <br />
The agarose was poured into the plate and cooled. In preparing the samples for the loading gel, 10µL of the YFP samples, 4µL of 6X DNA Loading dye and 10µL of distilled water were added to eppendorf tubes. 12µL of the samples were loaded onto the gel which was placed into the tank filled with TAE buffer. Electrophoresis was run at 120V for 30 min. <br />
<br />
<br />
|width="50%"|[[Image:SNC00109.jpg|300px|centre|Agarose gel prepared for the electrophoresis of YFP|thumb]]<br />
|}<br />
<br><br />
<br />
==Mutagenesis of YFP ==<br />
<br />
'''''Mutant Strand Synthesis Reaction'''''<br />
----<br />
<br />
<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|<br />
The mutagenesis of YFP was completed using polymerase chain reaction. The control reaction was prepared as indicated below:<br />
<br />
The YFP plasmid was diluted by a 1:17 dilution factor to obtain a concentration of 2.13 µL.<br />
|width="50%"| Lastly, 1µL of DNA polymerase was added. The reaction was done using 18 cycles with the following cycling parameters: <br />
|-<br />
|width="50%"|Primer 657 was diluted to a concentration of 24.1ng/µL using a 1:5 dilution and primer 656 was diluted to 27.1ng/µL using a 1:9 dilution. These dilutions were done to bring the concentrations of the plasmids to 125ng. <br />
Following the dilutions the following was carried out:<br />
<br />
5µL of 10X reaction buffer, 2.13µL YFP plasmid, 5.18µL 657 primer, 4.61µL 656 primer, 1µL of dNTP mix, 3µL of quick solution and 29.08µL of MQ water were added to a eppendorf tube making a total volume of 50µL. <br />
<br />
|width="50%" align="center"|<br />
{| class="wikitable" border="1"<br />
|-<br />
! Segments<br />
! Cycles<br />
! Temperature<br />
! Time<br />
|-<br />
| 1<br />
| 1<br />
| 95°C<br />
| 1 minute<br />
|-<br />
| 2<br />
| 18<br />
| 95°C<br />
| 50 seconds<br />
|-<br />
| <br />
| <br />
| 60°C<br />
| 50 seconds<br />
|-<br />
| <br />
| <br />
| 68°C<br />
| 1 minute<br />
|-<br />
| 3<br />
| 1<br />
| 68°C<br />
| 7 minutes<br />
|}<br />
<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''''Dpn 1 Digestion of the Amplification Product'''''<br />
----<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|1µL of the Dpn I restriction enyme at 10 U/µL was directly added the amplifictaion reaction in the tube and using a pipette it was thoroguhly mixed by pipetting the solution up and down several times. The reaction mixture was microcentrifuged<br />
|width="50%" | for 1 minute and incubated at 37°C for 7 hours to digest the parental supercoiled dsDNA.</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Project/Materials_and_MethodsTeam:Victoria Australia/Project/Materials and Methods2009-10-20T15:30:12Z<p>Muriel aldunate: /* Electrophoresis of YFP */</p>
<hr />
<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
<br />
'''''<br />
== <div style="width: 60%; height: 60%; color: #50cad; font-size: 25px; ">Materials & Methods</div> ==<br />
'''''<br />
<br />
__TOC__<br />
==Transformation of fluorescent cells==<br />
{|style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top"<br />
|width="50%" |To begin with, all of the required reagent solutions were prepared. LB Agar plates (protocol 1) were made using 10g tryptone digest of casein, 5g yeast extract, 5g NaCl, 15g agar and then made up to 1L using milliQ water. Ampicillin (100μg/ml) was then prepared by adding 1gram of ampicillin and 9 ml (9gram) milliQ water so that the total volume was 10ml. LB media was made using 10g tryptone, 5g yeast extract, 5g NaCl and milliQ water to make the media up to 1L. <br><br />
<br />
The bacterial transformation of both BFP and GFP was completed by firstly Pre-incubating the LB-agar plates at 37°C for 2 hours prior. The Ca-competent cells were thawed on ice (4ºC). <br />
|width="50%"|Plasmid DNA was added and the tubes stored on ice for 30mins. The tubes were transferred to a rack placed in a preheated water bath (45ºC) and incubated for 30secs. The tubes were then transferred to ice (4ºC) and incubated on ice for 2 min. 800μl of SOC medium was added to each tube and Incubated at 37ºC with gentle shaking (no more that 50rpm) for 45min. 50μl of the transformed cells was transferred onto the LB agar plates each containing ampicillin, and spread the cell suspension thoroughly on the surface using a sterile rod (Streaking). Plates were incubated (agar side up) at 37ºC for ~16h. <br />
Transformation of Vic GFP, BFP, YFP, AZ, blueberry and cherry was done using the same protocol as above. <br />
<br><br />
|}<br />
<br />
==Protein expression==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|Following the transformation, protein expression was carried out by adding 100ml of Lb media into two conical flasks that were previously autoclaved. Using a pipette tip a single colony from the transformed bacteria of GFP and BFP was selected and dropped into conical flask. They were then incubated at 240rpm at 38 ºC for 18 hours.<br />
Using the incubated GFP and BFP they were divided into two falcon tubes each and centrifuged at 3000rpm for 20min. The supernatant was emptied (waste) and the pellet was resuspended with 20ml of SOC solution and 20 µL of ampicillin being added. At a wavelength of 600nm, the solution was measured in the spectrometer to check the density of the bacteria.The results obtained were as follows: GFP = 0.93A and BFP = 0.95A.Two conical flasks were made <br />
|width="50%" |up with 120ml SOC solution and 120 µL of Ampicillin (this is the solution in which GFP and BFP are added to).<br />
Using C1V1=C2V2 the amount of GFP and BFP that was added to the solution from above (SOC and Ampicillin) was found.After adding the GFP/BFP the solution was incubated at 240rpm at 28 ºC for 1 hour. Following this hour the absorbance was read at 600nm and the following results were obtained: GFP = 0.7A (undiluted) and BFP = 0.64A (undiluted).<br />
The proteins were then induced by adding 147µL of IPTG into each conical flask. This was poured into 2 50 ml falcon tubes and centrifuged for 10 min at 2000rpm. Stored at -80°C. <br />
<br />
|}<br />
<br />
==Preparation of reagents==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer. <br />
|width="50%" |100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution. 1M imieazole was prepared by adding 0.6808g of imieazole to 10ml of milliQ water. <br />
|}<br />
==Purification of GFP and BFP==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or BFP. That is; GFP with lysis buffer added to pellet with GFP and BFP with lysis buffer added to pellet with BFP. These were sonicated to split the cells open at 70% duty cycle 30seconds on and 20seconds off for 3 cycles. Samples then centrifuged for 40min. The supernatant was transferred into eppendorf tubes (2 times GFP and 2times BFP). 250 µL of resin was added to each tube and placed on suspension mixer for 20min. again, the supernatant was removed and added 1ml phosphate buffer to the remaining resin. <br />
This was centrifuged at 500rpm for 2min and the supernatant removed with 1ml phosphate buffer being again added. This was centrifuged at 500rpm for 2min and the supernatant removed. The pellet was resuspended with 5ml elution buffer (An elution buffer was made using 2.5ml imieazole with phosphate buffer being added resulting in a total volume of 10ml. ) again this was centrifuged for 5min at 500rpm. The resulting supernatant formed contained the GFP and BFP; this was removed. <br />
<br />
<br />
|width="50%"|<br />
[[Image:SNC00184.jpg|300px|centre|Purified GFP and BFP obtained in the supernatant|thumb]]<br />
|}<br />
<br />
==Preparation of yellow plasmid with E coli bacteria==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|A 6M guanidine hydrochloride solution was prepared along with 50ml Tris Buffer at pH 7.8 ( 1M Tris-HCL with the pH adjusted by adding NaOH). 4M Guanidinium in mix with buffer was added to the protein solution and spun for 30 minutes. This was then centrifuged at 20233rcf and the supernatant put in eppendorf tubes with resin. The ampicillin was then incubated with 1 micro litre/ml working stock 100µg/ml. <br />
4ml of LB medium was put into a flacon tube. With a pipette tip E.coli bacteria was scraped and placed into the falcon tube. It was then incubated overnight at 37 ºC 300rpm. 100ml 2YT solution added to 4 conical flasks. 1ml of the incubated Bactria stock (from above) was put into each flask and incubated at 180rpm 37 ºC. <br />
|width="50%" |After incubation it was centrifuged at 2880rpm for 20min and the supernatant poured into a conical flask (2YT) with decon disinfectant to kill off any remaining bacteria. 8 falcon tubes with the bacteria pellet were then stored (frozen). 6ml of LB media was added to 4 times 15ml falcon tubes. 0.1µl/nl = 6C ampicillin was put in and a stab of yellow fluorescent protein (3.1) was then added to the solution and resuspended. This was incubated at 190rpm at 37 ºC for 16 hours. Following incubation, the solution was centrifuged at 21000rpm for 5 min.<br />
|}<br />
<br />
==Purification of yellow fluorescent plasmid==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|The aurum plasmid spin format protocol was completed (The plasmid containing bacterial host of the yellow protein was transferred to a 1.5-2.0ml capped microcentrifuge tube. Cells were pelleted for 1 min at full speed and the supernatant removed. 250µL of resuspension solution was added and pipetted up and down until the cell pellet was completely resuspended. 250µL of lysis solution was added and mixed by inverting. 5 min after the lysis was added, 350µL of neutralisation solution was added and also mixed by inversion. The neutralised lysate was centrifuged for 5min at 21000rpm {the supernatant or cleared lysate contains the plasmid DNA}.<br />
While centrifuging, a plasmid mini column was inserted into a 2ml capless wash tube. The supernatant was transferred to the plasmid mini column and centrifuged for 1min.<br />
|width="50%" |Wash solution at 5X concentrate was used to add 4 volumes (100ml) of 95-100% ethanol. The plasmid mini column from the wash tube was removed and the filtrate discarded from the tube. The column was replaced into the same wash tube. 750µL of the wash solution was added and centrifuged for 1min. The wash solution was discarded from the tube and the column was replaced into the same wash tube. It was then centrifuged for an additional 1 min to remove the residual wash solution. The plasmid mini column was transferred to a 1.5-2.0ml capped microcentrifuge tube and <br />
50µL of elution solution was added onto the membrane stack at the base of the column and allowed a minute for the solution to saturate the membranes. It was again centrifuged for 1min to elute the plasmid. The mini column was discarded and the eluted DNA was stored in the freezer).<br />
|}<br />
<br />
==Preparation of the E.coli cell free system==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|The E.Coli cell free system was constructed via the following method:<br />
Two buffers s30a and s30b were first made (1.725g potassium acetate, 2.944g magnesium sulphate, added 400ml MQ water and set to a pH of 7.8 by adding trisacetate. It was then filtered in to 2X 250ml beakers one being for S30A in which 500µL of 2 mercaptoethanol was added and S30B that did not contain 2 mercaptoehanol).<br />
The bacteria cells were thawed on ice and resuspended the pellets by washing out with S30A buffer and all he solution transferred into 2X falcon tubes. <br />
|width="50%" |These were then centrifuged at 2000 RCF for 10 minutes. Again, the pellets were resuspended with S30A buffer and centrifuged at 2000 RCF for 10 minutes. This was completed twice as to a total of 3 times. The supernatant was removed. The pellet was resuspended with S30B buffer and then sonicated for 30 sec on and 30 sec off at 50% for a total of 5 minutes. <br />
1ml samples were placed 10 eppendorf tubes and centrifuged at 20 000 for 30 minutes at 4°C. Supernatant was transferred into clean eppendorf tubes and also centrifuged at 20000 rpm for 45 minutes. 7.25ml of the supernatant was removed to serve as the cell lysate in the E.coli cell free system. <br />
|}<br />
<br />
==Expression and purification of yellow and green fluorescent protein==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|An expression and purification of the yellow and green fluorescent proteins was carried out to determine what cell line it had; DH5 alpha or BL21DE3.<br />
An overnight culture was set up in 2X falcon tubed with 5ml LB media and a pipette that was stabbed with YFP and GFP. 5µL of ampicillin was added and incubated overnight at 37°C at 220rpm. The protein expression was then completed by dividing the incubated YFP and GFP into 2X falcon tubes and centrifuged for 20 minutes at 3000rpm. <br />
The supernatant was emptied and the pellet resuspended with 20 ml of SOC solution and 20µL of ampicillin added. <br />
Spectrophotemetric analysis of the GFP and YFP at 1:100 dilution was done in the nanodrop 2000. <br />
|width="50%" |Absorbance at 600nm was taken before the dilution and the results were as follows: GFP=1.4 and YFP=0.8. the overnight cultures were then spun in the centrifuge to create a pellet for resuspension and again incubated at 37°C and 220rpm. The OD measurement was taken to be: YFP = 0.195 and GFP = 0.374.<br />
The proteins were then induced by adding 147µL of IPTG into each conical flask containing the YFP and GFP. This was poured into 2 x 50 ml falcon tubes, centrifuged for 10 min at 2000rpm and subsequently stored at -80°C.<br />
|}<br />
<br />
==Electrophoresis of YFP==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|To measure the purity in preparation for the electrophoresis run of the YFP protein, the following reagents were prepared: 6 X DNA gel loading dye (5.6ml of 80% glycerol, 9.2ml MQ water, 0.18ml EDTA at 0.5M and 0.018g bromphenol blue; made up to a volume of 15ml) and agarose gel (0.5g DNA grade agarose, 50µL TAE buffer, 2µL cybersafe to 20µL MQ water and 5µL of agarose gel). <br />
The agarose was poured into the plate and cooled. In preparing the samples for the loading gel, 10µL of the YFP samples, 4µL of 6X DNA Loading dye and 10µL of distilled water were added to eppendorf tubes. 12µL of the samples were loaded onto the gel which was placed into the tank filled with TAE buffer. Electrophoresis was run at 120V for 30 min. <br />
<br />
<br />
|width="50%"|[[Image:SNC00109.jpg|300px|centre|Agarose gel prepared for the electrophoresis of YFP|thumb]]<br />
|}<br />
<br><br />
<br />
==Mutagenesis of YFP ==<br />
<br />
'''''Mutant Strand Synthesis Reaction'''''<br />
----<br />
<br />
<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|<br />
The mutagenesis of YFP was completed using Polymerase chain reaction. The control reaction was prepared as indicated below:<br />
<br />
The YFP plasmid was diluted by a 1:17 dilution factor to obtain a concentration of 2.13 µL.<br />
|width="50%"| Lastly, 1µL of DNA polymerase was added. The reaction was done using 18 cycles with the following cycling parameters: <br />
|-<br />
|width="50%"|Primer 657 was diluted to a concentration of 24.1ng/µL using a 1:5 dilution and primer 656 was diluted to 27.1ng/µL using a 1:9 dilution. These dilutions were done to bring the concentrations of the plasmids to 125ng. <br />
Following the dilutions the following was carried out:<br />
<br />
5µL of 10X reaction buffer, 2.13µL YFP plasmid, 5.18µL 657 primer, 4.61µL 656 primer, 1µL of dNTP mix, 3µL of quick solution and 29.08µL of MQ water were added to a eppendorf tube making a total volume of 50µL. <br />
<br />
|width="50%" align="center"|<br />
{| class="wikitable" border="1"<br />
|-<br />
! Segments<br />
! Cycles<br />
! Temperature<br />
! Time<br />
|-<br />
| 1<br />
| 1<br />
| 95°C<br />
| 1 minute<br />
|-<br />
| 2<br />
| 18<br />
| 95°C<br />
| 50 seconds<br />
|-<br />
| <br />
| <br />
| 60°C<br />
| 50 seconds<br />
|-<br />
| <br />
| <br />
| 68°C<br />
| 1 minute<br />
|-<br />
| 3<br />
| 1<br />
| 68°C<br />
| 7 minutes<br />
|}<br />
<br />
|}<br />
<br />
'''''Dpn 1 Digestion of the Amplification Product'''''<br />
----<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|1µL of the Dpn I restriction enyme at 10 U/µL was directly added the amplifictaion reaction in the tube and using a pipette it was thoroguhly mixed by pipetting the solution up and down several times. The reaction mixture was microcentrifuged<br />
|width="50%" | for 1 minute and incubated at 37°C for 7 hours to digest the parental supercoiled dsDNA.</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Project/Materials_and_MethodsTeam:Victoria Australia/Project/Materials and Methods2009-10-20T15:24:07Z<p>Muriel aldunate: /* Electrophoresis of YFP */</p>
<hr />
<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
<br />
'''''<br />
== <div style="width: 60%; height: 60%; color: #50cad; font-size: 25px; ">Materials & Methods</div> ==<br />
'''''<br />
<br />
__TOC__<br />
==Transformation of fluorescent cells==<br />
{|style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top"<br />
|width="50%" |To begin with, all of the required reagent solutions were prepared. LB Agar plates (protocol 1) were made using 10g tryptone digest of casein, 5g yeast extract, 5g NaCl, 15g agar and then made up to 1L using milliQ water. Ampicillin (100μg/ml) was then prepared by adding 1gram of ampicillin and 9 ml (9gram) milliQ water so that the total volume was 10ml. LB media was made using 10g tryptone, 5g yeast extract, 5g NaCl and milliQ water to make the media up to 1L. <br><br />
<br />
The bacterial transformation of both BFP and GFP was completed by firstly Pre-incubating the LB-agar plates at 37°C for 2 hours prior. The Ca-competent cells were thawed on ice (4ºC). <br />
|width="50%"|Plasmid DNA was added and the tubes stored on ice for 30mins. The tubes were transferred to a rack placed in a preheated water bath (45ºC) and incubated for 30secs. The tubes were then transferred to ice (4ºC) and incubated on ice for 2 min. 800μl of SOC medium was added to each tube and Incubated at 37ºC with gentle shaking (no more that 50rpm) for 45min. 50μl of the transformed cells was transferred onto the LB agar plates each containing ampicillin, and spread the cell suspension thoroughly on the surface using a sterile rod (Streaking). Plates were incubated (agar side up) at 37ºC for ~16h. <br />
Transformation of Vic GFP, BFP, YFP, AZ, blueberry and cherry was done using the same protocol as above. <br />
<br><br />
|}<br />
<br />
==Protein expression==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|Following the transformation, protein expression was carried out by adding 100ml of Lb media into two conical flasks that were previously autoclaved. Using a pipette tip a single colony from the transformed bacteria of GFP and BFP was selected and dropped into conical flask. They were then incubated at 240rpm at 38 ºC for 18 hours.<br />
Using the incubated GFP and BFP they were divided into two falcon tubes each and centrifuged at 3000rpm for 20min. The supernatant was emptied (waste) and the pellet was resuspended with 20ml of SOC solution and 20 µL of ampicillin being added. At a wavelength of 600nm, the solution was measured in the spectrometer to check the density of the bacteria.The results obtained were as follows: GFP = 0.93A and BFP = 0.95A.Two conical flasks were made <br />
|width="50%" |up with 120ml SOC solution and 120 µL of Ampicillin (this is the solution in which GFP and BFP are added to).<br />
Using C1V1=C2V2 the amount of GFP and BFP that was added to the solution from above (SOC and Ampicillin) was found.After adding the GFP/BFP the solution was incubated at 240rpm at 28 ºC for 1 hour. Following this hour the absorbance was read at 600nm and the following results were obtained: GFP = 0.7A (undiluted) and BFP = 0.64A (undiluted).<br />
The proteins were then induced by adding 147µL of IPTG into each conical flask. This was poured into 2 50 ml falcon tubes and centrifuged for 10 min at 2000rpm. Stored at -80°C. <br />
<br />
|}<br />
<br />
==Preparation of reagents==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer. <br />
|width="50%" |100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution. 1M imieazole was prepared by adding 0.6808g of imieazole to 10ml of milliQ water. <br />
|}<br />
==Purification of GFP and BFP==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or BFP. That is; GFP with lysis buffer added to pellet with GFP and BFP with lysis buffer added to pellet with BFP. These were sonicated to split the cells open at 70% duty cycle 30seconds on and 20seconds off for 3 cycles. Samples then centrifuged for 40min. The supernatant was transferred into eppendorf tubes (2 times GFP and 2times BFP). 250 µL of resin was added to each tube and placed on suspension mixer for 20min. again, the supernatant was removed and added 1ml phosphate buffer to the remaining resin. <br />
This was centrifuged at 500rpm for 2min and the supernatant removed with 1ml phosphate buffer being again added. This was centrifuged at 500rpm for 2min and the supernatant removed. The pellet was resuspended with 5ml elution buffer (An elution buffer was made using 2.5ml imieazole with phosphate buffer being added resulting in a total volume of 10ml. ) again this was centrifuged for 5min at 500rpm. The resulting supernatant formed contained the GFP and BFP; this was removed. <br />
<br />
<br />
|width="50%"|<br />
[[Image:SNC00184.jpg|300px|centre|Purified GFP and BFP obtained in the supernatant|thumb]]<br />
|}<br />
<br />
==Preparation of yellow plasmid with E coli bacteria==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|A 6M guanidine hydrochloride solution was prepared along with 50ml Tris Buffer at pH 7.8 ( 1M Tris-HCL with the pH adjusted by adding NaOH). 4M Guanidinium in mix with buffer was added to the protein solution and spun for 30 minutes. This was then centrifuged at 20233rcf and the supernatant put in eppendorf tubes with resin. The ampicillin was then incubated with 1 micro litre/ml working stock 100µg/ml. <br />
4ml of LB medium was put into a flacon tube. With a pipette tip E.coli bacteria was scraped and placed into the falcon tube. It was then incubated overnight at 37 ºC 300rpm. 100ml 2YT solution added to 4 conical flasks. 1ml of the incubated Bactria stock (from above) was put into each flask and incubated at 180rpm 37 ºC. <br />
|width="50%" |After incubation it was centrifuged at 2880rpm for 20min and the supernatant poured into a conical flask (2YT) with decon disinfectant to kill off any remaining bacteria. 8 falcon tubes with the bacteria pellet were then stored (frozen). 6ml of LB media was added to 4 times 15ml falcon tubes. 0.1µl/nl = 6C ampicillin was put in and a stab of yellow fluorescent protein (3.1) was then added to the solution and resuspended. This was incubated at 190rpm at 37 ºC for 16 hours. Following incubation, the solution was centrifuged at 21000rpm for 5 min.<br />
|}<br />
<br />
==Purification of yellow fluorescent plasmid==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|The aurum plasmid spin format protocol was completed (The plasmid containing bacterial host of the yellow protein was transferred to a 1.5-2.0ml capped microcentrifuge tube. Cells were pelleted for 1 min at full speed and the supernatant removed. 250µL of resuspension solution was added and pipetted up and down until the cell pellet was completely resuspended. 250µL of lysis solution was added and mixed by inverting. 5 min after the lysis was added, 350µL of neutralisation solution was added and also mixed by inversion. The neutralised lysate was centrifuged for 5min at 21000rpm {the supernatant or cleared lysate contains the plasmid DNA}.<br />
While centrifuging, a plasmid mini column was inserted into a 2ml capless wash tube. The supernatant was transferred to the plasmid mini column and centrifuged for 1min.<br />
|width="50%" |Wash solution at 5X concentrate was used to add 4 volumes (100ml) of 95-100% ethanol. The plasmid mini column from the wash tube was removed and the filtrate discarded from the tube. The column was replaced into the same wash tube. 750µL of the wash solution was added and centrifuged for 1min. The wash solution was discarded from the tube and the column was replaced into the same wash tube. It was then centrifuged for an additional 1 min to remove the residual wash solution. The plasmid mini column was transferred to a 1.5-2.0ml capped microcentrifuge tube and <br />
50µL of elution solution was added onto the membrane stack at the base of the column and allowed a minute for the solution to saturate the membranes. It was again centrifuged for 1min to elute the plasmid. The mini column was discarded and the eluted DNA was stored in the freezer).<br />
|}<br />
<br />
==Preparation of the E.coli cell free system==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|The E.Coli cell free system was constructed via the following method:<br />
Two buffers s30a and s30b were first made (1.725g potassium acetate, 2.944g magnesium sulphate, added 400ml MQ water and set to a pH of 7.8 by adding trisacetate. It was then filtered in to 2X 250ml beakers one being for S30A in which 500µL of 2 mercaptoethanol was added and S30B that did not contain 2 mercaptoehanol).<br />
The bacteria cells were thawed on ice and resuspended the pellets by washing out with S30A buffer and all he solution transferred into 2X falcon tubes. <br />
|width="50%" |These were then centrifuged at 2000 RCF for 10 minutes. Again, the pellets were resuspended with S30A buffer and centrifuged at 2000 RCF for 10 minutes. This was completed twice as to a total of 3 times. The supernatant was removed. The pellet was resuspended with S30B buffer and then sonicated for 30 sec on and 30 sec off at 50% for a total of 5 minutes. <br />
1ml samples were placed 10 eppendorf tubes and centrifuged at 20 000 for 30 minutes at 4°C. Supernatant was transferred into clean eppendorf tubes and also centrifuged at 20000 rpm for 45 minutes. 7.25ml of the supernatant was removed to serve as the cell lysate in the E.coli cell free system. <br />
|}<br />
<br />
==Expression and purification of yellow and green fluorescent protein==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|An expression and purification of the yellow and green fluorescent proteins was carried out to determine what cell line it had; DH5 alpha or BL21DE3.<br />
An overnight culture was set up in 2X falcon tubed with 5ml LB media and a pipette that was stabbed with YFP and GFP. 5µL of ampicillin was added and incubated overnight at 37°C at 220rpm. The protein expression was then completed by dividing the incubated YFP and GFP into 2X falcon tubes and centrifuged for 20 minutes at 3000rpm. <br />
The supernatant was emptied and the pellet resuspended with 20 ml of SOC solution and 20µL of ampicillin added. <br />
Spectrophotemetric analysis of the GFP and YFP at 1:100 dilution was done in the nanodrop 2000. <br />
|width="50%" |Absorbance at 600nm was taken before the dilution and the results were as follows: GFP=1.4 and YFP=0.8. the overnight cultures were then spun in the centrifuge to create a pellet for resuspension and again incubated at 37°C and 220rpm. The OD measurement was taken to be: YFP = 0.195 and GFP = 0.374.<br />
The proteins were then induced by adding 147µL of IPTG into each conical flask containing the YFP and GFP. This was poured into 2 x 50 ml falcon tubes, centrifuged for 10 min at 2000rpm and subsequently stored at -80°C.<br />
|}<br />
<br />
==Electrophoresis of YFP==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|To measure the purity in preparation for the electrophoresis run of the YFP protein, the following reagents were prepared: 6 X DNA gel loading dye (5.6ml of 80% glycerol, 9.2ml MQ water, 0.18ml EDTA at 0.5M and 0.018g bromphenol blue; made up to a volume of 15ml) and agarose gel (0.5g DNA grade agarose, 50µL TAE buffer, 2µL cybersafe to 20µL MQ water and 5µL of agarose gel). <br />
The agarose was poured into the plate and cooled. In preparing the samples for the loading gel, 10µL of the YFP samples, 4µL of 6X DNA Loading dye and 10µL of distilled water were added to eppendorf tubes. 12µL of the samples were loaded onto the gel which was placed into the tank filled with TAE buffer. Electrophoresis was run at 120V for 30 min. <br />
<br />
<br />
|width="50%"|[[Image:SNC00109.jpg|300px|centre|thumb]]<br />
|}<br />
<br><br />
<br />
==Mutagenesis of YFP ==<br />
<br />
'''''Mutant Strand Synthesis Reaction'''''<br />
----<br />
<br />
<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|<br />
The mutagenesis of YFP was completed using Polymerase chain reaction. The control reaction was prepared as indicated below:<br />
<br />
The YFP plasmid was diluted by a 1:17 dilution factor to obtain a concentration of 2.13 µL.<br />
|width="50%"| Lastly, 1µL of DNA polymerase was added. The reaction was done using 18 cycles with the following cycling parameters: <br />
|-<br />
|width="50%"|Primer 657 was diluted to a concentration of 24.1ng/µL using a 1:5 dilution and primer 656 was diluted to 27.1ng/µL using a 1:9 dilution. These dilutions were done to bring the concentrations of the plasmids to 125ng. <br />
Following the dilutions the following was carried out:<br />
<br />
5µL of 10X reaction buffer, 2.13µL YFP plasmid, 5.18µL 657 primer, 4.61µL 656 primer, 1µL of dNTP mix, 3µL of quick solution and 29.08µL of MQ water were added to a eppendorf tube making a total volume of 50µL. <br />
<br />
|width="50%" align="center"|<br />
{| class="wikitable" border="1"<br />
|-<br />
! Segments<br />
! Cycles<br />
! Temperature<br />
! Time<br />
|-<br />
| 1<br />
| 1<br />
| 95°C<br />
| 1 minute<br />
|-<br />
| 2<br />
| 18<br />
| 95°C<br />
| 50 seconds<br />
|-<br />
| <br />
| <br />
| 60°C<br />
| 50 seconds<br />
|-<br />
| <br />
| <br />
| 68°C<br />
| 1 minute<br />
|-<br />
| 3<br />
| 1<br />
| 68°C<br />
| 7 minutes<br />
|}<br />
<br />
|}<br />
<br />
'''''Dpn 1 Digestion of the Amplification Product'''''<br />
----<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|1µL of the Dpn I restriction enyme at 10 U/µL was directly added the amplifictaion reaction in the tube and using a pipette it was thoroguhly mixed by pipetting the solution up and down several times. The reaction mixture was microcentrifuged<br />
|width="50%" | for 1 minute and incubated at 37°C for 7 hours to digest the parental supercoiled dsDNA.</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Project/Materials_and_MethodsTeam:Victoria Australia/Project/Materials and Methods2009-10-20T15:20:07Z<p>Muriel aldunate: /* Expression and purification of yellow and green fluorescent protein */</p>
<hr />
<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
<br />
'''''<br />
== <div style="width: 60%; height: 60%; color: #50cad; font-size: 25px; ">Materials & Methods</div> ==<br />
'''''<br />
<br />
__TOC__<br />
==Transformation of fluorescent cells==<br />
{|style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top"<br />
|width="50%" |To begin with, all of the required reagent solutions were prepared. LB Agar plates (protocol 1) were made using 10g tryptone digest of casein, 5g yeast extract, 5g NaCl, 15g agar and then made up to 1L using milliQ water. Ampicillin (100μg/ml) was then prepared by adding 1gram of ampicillin and 9 ml (9gram) milliQ water so that the total volume was 10ml. LB media was made using 10g tryptone, 5g yeast extract, 5g NaCl and milliQ water to make the media up to 1L. <br><br />
<br />
The bacterial transformation of both BFP and GFP was completed by firstly Pre-incubating the LB-agar plates at 37°C for 2 hours prior. The Ca-competent cells were thawed on ice (4ºC). <br />
|width="50%"|Plasmid DNA was added and the tubes stored on ice for 30mins. The tubes were transferred to a rack placed in a preheated water bath (45ºC) and incubated for 30secs. The tubes were then transferred to ice (4ºC) and incubated on ice for 2 min. 800μl of SOC medium was added to each tube and Incubated at 37ºC with gentle shaking (no more that 50rpm) for 45min. 50μl of the transformed cells was transferred onto the LB agar plates each containing ampicillin, and spread the cell suspension thoroughly on the surface using a sterile rod (Streaking). Plates were incubated (agar side up) at 37ºC for ~16h. <br />
Transformation of Vic GFP, BFP, YFP, AZ, blueberry and cherry was done using the same protocol as above. <br />
<br><br />
|}<br />
<br />
==Protein expression==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|Following the transformation, protein expression was carried out by adding 100ml of Lb media into two conical flasks that were previously autoclaved. Using a pipette tip a single colony from the transformed bacteria of GFP and BFP was selected and dropped into conical flask. They were then incubated at 240rpm at 38 ºC for 18 hours.<br />
Using the incubated GFP and BFP they were divided into two falcon tubes each and centrifuged at 3000rpm for 20min. The supernatant was emptied (waste) and the pellet was resuspended with 20ml of SOC solution and 20 µL of ampicillin being added. At a wavelength of 600nm, the solution was measured in the spectrometer to check the density of the bacteria.The results obtained were as follows: GFP = 0.93A and BFP = 0.95A.Two conical flasks were made <br />
|width="50%" |up with 120ml SOC solution and 120 µL of Ampicillin (this is the solution in which GFP and BFP are added to).<br />
Using C1V1=C2V2 the amount of GFP and BFP that was added to the solution from above (SOC and Ampicillin) was found.After adding the GFP/BFP the solution was incubated at 240rpm at 28 ºC for 1 hour. Following this hour the absorbance was read at 600nm and the following results were obtained: GFP = 0.7A (undiluted) and BFP = 0.64A (undiluted).<br />
The proteins were then induced by adding 147µL of IPTG into each conical flask. This was poured into 2 50 ml falcon tubes and centrifuged for 10 min at 2000rpm. Stored at -80°C. <br />
<br />
|}<br />
<br />
==Preparation of reagents==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer. <br />
|width="50%" |100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution. 1M imieazole was prepared by adding 0.6808g of imieazole to 10ml of milliQ water. <br />
|}<br />
==Purification of GFP and BFP==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or BFP. That is; GFP with lysis buffer added to pellet with GFP and BFP with lysis buffer added to pellet with BFP. These were sonicated to split the cells open at 70% duty cycle 30seconds on and 20seconds off for 3 cycles. Samples then centrifuged for 40min. The supernatant was transferred into eppendorf tubes (2 times GFP and 2times BFP). 250 µL of resin was added to each tube and placed on suspension mixer for 20min. again, the supernatant was removed and added 1ml phosphate buffer to the remaining resin. <br />
This was centrifuged at 500rpm for 2min and the supernatant removed with 1ml phosphate buffer being again added. This was centrifuged at 500rpm for 2min and the supernatant removed. The pellet was resuspended with 5ml elution buffer (An elution buffer was made using 2.5ml imieazole with phosphate buffer being added resulting in a total volume of 10ml. ) again this was centrifuged for 5min at 500rpm. The resulting supernatant formed contained the GFP and BFP; this was removed. <br />
<br />
<br />
|width="50%"|<br />
[[Image:SNC00184.jpg|300px|centre|Purified GFP and BFP obtained in the supernatant|thumb]]<br />
|}<br />
<br />
==Preparation of yellow plasmid with E coli bacteria==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|A 6M guanidine hydrochloride solution was prepared along with 50ml Tris Buffer at pH 7.8 ( 1M Tris-HCL with the pH adjusted by adding NaOH). 4M Guanidinium in mix with buffer was added to the protein solution and spun for 30 minutes. This was then centrifuged at 20233rcf and the supernatant put in eppendorf tubes with resin. The ampicillin was then incubated with 1 micro litre/ml working stock 100µg/ml. <br />
4ml of LB medium was put into a flacon tube. With a pipette tip E.coli bacteria was scraped and placed into the falcon tube. It was then incubated overnight at 37 ºC 300rpm. 100ml 2YT solution added to 4 conical flasks. 1ml of the incubated Bactria stock (from above) was put into each flask and incubated at 180rpm 37 ºC. <br />
|width="50%" |After incubation it was centrifuged at 2880rpm for 20min and the supernatant poured into a conical flask (2YT) with decon disinfectant to kill off any remaining bacteria. 8 falcon tubes with the bacteria pellet were then stored (frozen). 6ml of LB media was added to 4 times 15ml falcon tubes. 0.1µl/nl = 6C ampicillin was put in and a stab of yellow fluorescent protein (3.1) was then added to the solution and resuspended. This was incubated at 190rpm at 37 ºC for 16 hours. Following incubation, the solution was centrifuged at 21000rpm for 5 min.<br />
|}<br />
<br />
==Purification of yellow fluorescent plasmid==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|The aurum plasmid spin format protocol was completed (The plasmid containing bacterial host of the yellow protein was transferred to a 1.5-2.0ml capped microcentrifuge tube. Cells were pelleted for 1 min at full speed and the supernatant removed. 250µL of resuspension solution was added and pipetted up and down until the cell pellet was completely resuspended. 250µL of lysis solution was added and mixed by inverting. 5 min after the lysis was added, 350µL of neutralisation solution was added and also mixed by inversion. The neutralised lysate was centrifuged for 5min at 21000rpm {the supernatant or cleared lysate contains the plasmid DNA}.<br />
While centrifuging, a plasmid mini column was inserted into a 2ml capless wash tube. The supernatant was transferred to the plasmid mini column and centrifuged for 1min.<br />
|width="50%" |Wash solution at 5X concentrate was used to add 4 volumes (100ml) of 95-100% ethanol. The plasmid mini column from the wash tube was removed and the filtrate discarded from the tube. The column was replaced into the same wash tube. 750µL of the wash solution was added and centrifuged for 1min. The wash solution was discarded from the tube and the column was replaced into the same wash tube. It was then centrifuged for an additional 1 min to remove the residual wash solution. The plasmid mini column was transferred to a 1.5-2.0ml capped microcentrifuge tube and <br />
50µL of elution solution was added onto the membrane stack at the base of the column and allowed a minute for the solution to saturate the membranes. It was again centrifuged for 1min to elute the plasmid. The mini column was discarded and the eluted DNA was stored in the freezer).<br />
|}<br />
<br />
==Preparation of the E.coli cell free system==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|The E.Coli cell free system was constructed via the following method:<br />
Two buffers s30a and s30b were first made (1.725g potassium acetate, 2.944g magnesium sulphate, added 400ml MQ water and set to a pH of 7.8 by adding trisacetate. It was then filtered in to 2X 250ml beakers one being for S30A in which 500µL of 2 mercaptoethanol was added and S30B that did not contain 2 mercaptoehanol).<br />
The bacteria cells were thawed on ice and resuspended the pellets by washing out with S30A buffer and all he solution transferred into 2X falcon tubes. <br />
|width="50%" |These were then centrifuged at 2000 RCF for 10 minutes. Again, the pellets were resuspended with S30A buffer and centrifuged at 2000 RCF for 10 minutes. This was completed twice as to a total of 3 times. The supernatant was removed. The pellet was resuspended with S30B buffer and then sonicated for 30 sec on and 30 sec off at 50% for a total of 5 minutes. <br />
1ml samples were placed 10 eppendorf tubes and centrifuged at 20 000 for 30 minutes at 4°C. Supernatant was transferred into clean eppendorf tubes and also centrifuged at 20000 rpm for 45 minutes. 7.25ml of the supernatant was removed to serve as the cell lysate in the E.coli cell free system. <br />
|}<br />
<br />
==Expression and purification of yellow and green fluorescent protein==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|An expression and purification of the yellow and green fluorescent proteins was carried out to determine what cell line it had; DH5 alpha or BL21DE3.<br />
An overnight culture was set up in 2X falcon tubed with 5ml LB media and a pipette that was stabbed with YFP and GFP. 5µL of ampicillin was added and incubated overnight at 37°C at 220rpm. The protein expression was then completed by dividing the incubated YFP and GFP into 2X falcon tubes and centrifuged for 20 minutes at 3000rpm. <br />
The supernatant was emptied and the pellet resuspended with 20 ml of SOC solution and 20µL of ampicillin added. <br />
Spectrophotemetric analysis of the GFP and YFP at 1:100 dilution was done in the nanodrop 2000. <br />
|width="50%" |Absorbance at 600nm was taken before the dilution and the results were as follows: GFP=1.4 and YFP=0.8. the overnight cultures were then spun in the centrifuge to create a pellet for resuspension and again incubated at 37°C and 220rpm. The OD measurement was taken to be: YFP = 0.195 and GFP = 0.374.<br />
The proteins were then induced by adding 147µL of IPTG into each conical flask containing the YFP and GFP. This was poured into 2 x 50 ml falcon tubes, centrifuged for 10 min at 2000rpm and subsequently stored at -80°C.<br />
|}<br />
<br />
==Electrophoresis of YFP==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|In preparation for the electrophoresis run of the YFP protein to measure its purity, reagents were made being: 6X DNA gel loading dye (5.6ml of 80% glycerol, 9.2ml MQ water, 0.18ml EDTA at 0.5M and 0.018g bromphenol blue. This wade up to a volume of 15ml) and agarose gel (0.5g DNA grade agarose, 50µL TAE buffer, 2µL cybersafe to 20µL MQ water and 5µL of agarose gel). <br />
The agarose was poured into the plate and cooled. In preparing the samples for the loading gel, 10µL of the YFP samples, 4µL of 6X DNA Loading dye and 10µL of distilled water were added to eppendorf tubes. 12µL of the samples were loaded onto the gel. The plate was put into the tank that was filled with TAE buffer. Electrophoresis was run at 120V for 30 min. <br />
<br />
<br />
|width="50%"|[[Image:SNC00109.jpg|300px|centre|thumb]]<br />
|}<br />
<br><br />
<br />
==Mutagenesis of YFP ==<br />
<br />
'''''Mutant Strand Synthesis Reaction'''''<br />
----<br />
<br />
<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|<br />
The mutagenesis of YFP was completed using Polymerase chain reaction. The control reaction was prepared as indicated below:<br />
<br />
The YFP plasmid was diluted by a 1:17 dilution factor to obtain a concentration of 2.13 µL.<br />
|width="50%"| Lastly, 1µL of DNA polymerase was added. The reaction was done using 18 cycles with the following cycling parameters: <br />
|-<br />
|width="50%"|Primer 657 was diluted to a concentration of 24.1ng/µL using a 1:5 dilution and primer 656 was diluted to 27.1ng/µL using a 1:9 dilution. These dilutions were done to bring the concentrations of the plasmids to 125ng. <br />
Following the dilutions the following was carried out:<br />
<br />
5µL of 10X reaction buffer, 2.13µL YFP plasmid, 5.18µL 657 primer, 4.61µL 656 primer, 1µL of dNTP mix, 3µL of quick solution and 29.08µL of MQ water were added to a eppendorf tube making a total volume of 50µL. <br />
<br />
|width="50%" align="center"|<br />
{| class="wikitable" border="1"<br />
|-<br />
! Segments<br />
! Cycles<br />
! Temperature<br />
! Time<br />
|-<br />
| 1<br />
| 1<br />
| 95°C<br />
| 1 minute<br />
|-<br />
| 2<br />
| 18<br />
| 95°C<br />
| 50 seconds<br />
|-<br />
| <br />
| <br />
| 60°C<br />
| 50 seconds<br />
|-<br />
| <br />
| <br />
| 68°C<br />
| 1 minute<br />
|-<br />
| 3<br />
| 1<br />
| 68°C<br />
| 7 minutes<br />
|}<br />
<br />
|}<br />
<br />
'''''Dpn 1 Digestion of the Amplification Product'''''<br />
----<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|1µL of the Dpn I restriction enyme at 10 U/µL was directly added the amplifictaion reaction in the tube and using a pipette it was thoroguhly mixed by pipetting the solution up and down several times. The reaction mixture was microcentrifuged<br />
|width="50%" | for 1 minute and incubated at 37°C for 7 hours to digest the parental supercoiled dsDNA.</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Project/Materials_and_MethodsTeam:Victoria Australia/Project/Materials and Methods2009-10-20T15:18:58Z<p>Muriel aldunate: /* Preparation of the E.coli cell free system */</p>
<hr />
<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
<br />
'''''<br />
== <div style="width: 60%; height: 60%; color: #50cad; font-size: 25px; ">Materials & Methods</div> ==<br />
'''''<br />
<br />
__TOC__<br />
==Transformation of fluorescent cells==<br />
{|style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top"<br />
|width="50%" |To begin with, all of the required reagent solutions were prepared. LB Agar plates (protocol 1) were made using 10g tryptone digest of casein, 5g yeast extract, 5g NaCl, 15g agar and then made up to 1L using milliQ water. Ampicillin (100μg/ml) was then prepared by adding 1gram of ampicillin and 9 ml (9gram) milliQ water so that the total volume was 10ml. LB media was made using 10g tryptone, 5g yeast extract, 5g NaCl and milliQ water to make the media up to 1L. <br><br />
<br />
The bacterial transformation of both BFP and GFP was completed by firstly Pre-incubating the LB-agar plates at 37°C for 2 hours prior. The Ca-competent cells were thawed on ice (4ºC). <br />
|width="50%"|Plasmid DNA was added and the tubes stored on ice for 30mins. The tubes were transferred to a rack placed in a preheated water bath (45ºC) and incubated for 30secs. The tubes were then transferred to ice (4ºC) and incubated on ice for 2 min. 800μl of SOC medium was added to each tube and Incubated at 37ºC with gentle shaking (no more that 50rpm) for 45min. 50μl of the transformed cells was transferred onto the LB agar plates each containing ampicillin, and spread the cell suspension thoroughly on the surface using a sterile rod (Streaking). Plates were incubated (agar side up) at 37ºC for ~16h. <br />
Transformation of Vic GFP, BFP, YFP, AZ, blueberry and cherry was done using the same protocol as above. <br />
<br><br />
|}<br />
<br />
==Protein expression==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|Following the transformation, protein expression was carried out by adding 100ml of Lb media into two conical flasks that were previously autoclaved. Using a pipette tip a single colony from the transformed bacteria of GFP and BFP was selected and dropped into conical flask. They were then incubated at 240rpm at 38 ºC for 18 hours.<br />
Using the incubated GFP and BFP they were divided into two falcon tubes each and centrifuged at 3000rpm for 20min. The supernatant was emptied (waste) and the pellet was resuspended with 20ml of SOC solution and 20 µL of ampicillin being added. At a wavelength of 600nm, the solution was measured in the spectrometer to check the density of the bacteria.The results obtained were as follows: GFP = 0.93A and BFP = 0.95A.Two conical flasks were made <br />
|width="50%" |up with 120ml SOC solution and 120 µL of Ampicillin (this is the solution in which GFP and BFP are added to).<br />
Using C1V1=C2V2 the amount of GFP and BFP that was added to the solution from above (SOC and Ampicillin) was found.After adding the GFP/BFP the solution was incubated at 240rpm at 28 ºC for 1 hour. Following this hour the absorbance was read at 600nm and the following results were obtained: GFP = 0.7A (undiluted) and BFP = 0.64A (undiluted).<br />
The proteins were then induced by adding 147µL of IPTG into each conical flask. This was poured into 2 50 ml falcon tubes and centrifuged for 10 min at 2000rpm. Stored at -80°C. <br />
<br />
|}<br />
<br />
==Preparation of reagents==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer. <br />
|width="50%" |100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution. 1M imieazole was prepared by adding 0.6808g of imieazole to 10ml of milliQ water. <br />
|}<br />
==Purification of GFP and BFP==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or BFP. That is; GFP with lysis buffer added to pellet with GFP and BFP with lysis buffer added to pellet with BFP. These were sonicated to split the cells open at 70% duty cycle 30seconds on and 20seconds off for 3 cycles. Samples then centrifuged for 40min. The supernatant was transferred into eppendorf tubes (2 times GFP and 2times BFP). 250 µL of resin was added to each tube and placed on suspension mixer for 20min. again, the supernatant was removed and added 1ml phosphate buffer to the remaining resin. <br />
This was centrifuged at 500rpm for 2min and the supernatant removed with 1ml phosphate buffer being again added. This was centrifuged at 500rpm for 2min and the supernatant removed. The pellet was resuspended with 5ml elution buffer (An elution buffer was made using 2.5ml imieazole with phosphate buffer being added resulting in a total volume of 10ml. ) again this was centrifuged for 5min at 500rpm. The resulting supernatant formed contained the GFP and BFP; this was removed. <br />
<br />
<br />
|width="50%"|<br />
[[Image:SNC00184.jpg|300px|centre|Purified GFP and BFP obtained in the supernatant|thumb]]<br />
|}<br />
<br />
==Preparation of yellow plasmid with E coli bacteria==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|A 6M guanidine hydrochloride solution was prepared along with 50ml Tris Buffer at pH 7.8 ( 1M Tris-HCL with the pH adjusted by adding NaOH). 4M Guanidinium in mix with buffer was added to the protein solution and spun for 30 minutes. This was then centrifuged at 20233rcf and the supernatant put in eppendorf tubes with resin. The ampicillin was then incubated with 1 micro litre/ml working stock 100µg/ml. <br />
4ml of LB medium was put into a flacon tube. With a pipette tip E.coli bacteria was scraped and placed into the falcon tube. It was then incubated overnight at 37 ºC 300rpm. 100ml 2YT solution added to 4 conical flasks. 1ml of the incubated Bactria stock (from above) was put into each flask and incubated at 180rpm 37 ºC. <br />
|width="50%" |After incubation it was centrifuged at 2880rpm for 20min and the supernatant poured into a conical flask (2YT) with decon disinfectant to kill off any remaining bacteria. 8 falcon tubes with the bacteria pellet were then stored (frozen). 6ml of LB media was added to 4 times 15ml falcon tubes. 0.1µl/nl = 6C ampicillin was put in and a stab of yellow fluorescent protein (3.1) was then added to the solution and resuspended. This was incubated at 190rpm at 37 ºC for 16 hours. Following incubation, the solution was centrifuged at 21000rpm for 5 min.<br />
|}<br />
<br />
==Purification of yellow fluorescent plasmid==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|The aurum plasmid spin format protocol was completed (The plasmid containing bacterial host of the yellow protein was transferred to a 1.5-2.0ml capped microcentrifuge tube. Cells were pelleted for 1 min at full speed and the supernatant removed. 250µL of resuspension solution was added and pipetted up and down until the cell pellet was completely resuspended. 250µL of lysis solution was added and mixed by inverting. 5 min after the lysis was added, 350µL of neutralisation solution was added and also mixed by inversion. The neutralised lysate was centrifuged for 5min at 21000rpm {the supernatant or cleared lysate contains the plasmid DNA}.<br />
While centrifuging, a plasmid mini column was inserted into a 2ml capless wash tube. The supernatant was transferred to the plasmid mini column and centrifuged for 1min.<br />
|width="50%" |Wash solution at 5X concentrate was used to add 4 volumes (100ml) of 95-100% ethanol. The plasmid mini column from the wash tube was removed and the filtrate discarded from the tube. The column was replaced into the same wash tube. 750µL of the wash solution was added and centrifuged for 1min. The wash solution was discarded from the tube and the column was replaced into the same wash tube. It was then centrifuged for an additional 1 min to remove the residual wash solution. The plasmid mini column was transferred to a 1.5-2.0ml capped microcentrifuge tube and <br />
50µL of elution solution was added onto the membrane stack at the base of the column and allowed a minute for the solution to saturate the membranes. It was again centrifuged for 1min to elute the plasmid. The mini column was discarded and the eluted DNA was stored in the freezer).<br />
|}<br />
<br />
==Preparation of the E.coli cell free system==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|The E.Coli cell free system was constructed via the following method:<br />
Two buffers s30a and s30b were first made (1.725g potassium acetate, 2.944g magnesium sulphate, added 400ml MQ water and set to a pH of 7.8 by adding trisacetate. It was then filtered in to 2X 250ml beakers one being for S30A in which 500µL of 2 mercaptoethanol was added and S30B that did not contain 2 mercaptoehanol).<br />
The bacteria cells were thawed on ice and resuspended the pellets by washing out with S30A buffer and all he solution transferred into 2X falcon tubes. <br />
|width="50%" |These were then centrifuged at 2000 RCF for 10 minutes. Again, the pellets were resuspended with S30A buffer and centrifuged at 2000 RCF for 10 minutes. This was completed twice as to a total of 3 times. The supernatant was removed. The pellet was resuspended with S30B buffer and then sonicated for 30 sec on and 30 sec off at 50% for a total of 5 minutes. <br />
1ml samples were placed 10 eppendorf tubes and centrifuged at 20 000 for 30 minutes at 4°C. Supernatant was transferred into clean eppendorf tubes and also centrifuged at 20000 rpm for 45 minutes. 7.25ml of the supernatant was removed to serve as the cell lysate in the E.coli cell free system. <br />
|}<br />
<br />
==Expression and purification of yellow and green fluorescent protein==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|An expression and purification of the yellow and green fluorescent proteins was carried out to determine what cell line it had; DH5 alpha or BL21DE3.<br />
An overnight culture was set up in 2X falcon tubed with 5ml LB media and a pipette that was stabbed with YFP and GFP. 5µL of ampicillin was added and incubated overnight at 37°C at 220rpm. The protein expression was then completed by dividing the incubated YFP and GFP into 2X falcon tubes and centrifuged for 20 minutes at 3000rpm. <br />
The supernatant was emptied and the pellet resuspended with 20 ml of SOC solution and 20µL of ampicillin added. <br />
Spectrophotemetric analysis of the GFP and YFP at 1:100 dilution was done in the nanodrop 2000. <br />
|width="50%" |Absorbance at 600nm was taken before the dilution and the results were as follows: GFP=1.4 and YFP=0.8. the overnight cultures were then spun in the centrifuge to create a pellet for resuspension and again incubated at 37°C and 220rpm. The OD measurement was taken to be: YFP = 0.195 and GFP = 0.374.<br />
The proteins were then induced by adding 147µL of IPTG into each conical flask containing the YFP and GFP. This was poured into 2 50 ml falcon tubes and centrifuged for 10 min at 2000rpm. Stored at -80°C.<br />
|}<br />
<br />
==Electrophoresis of YFP==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|In preparation for the electrophoresis run of the YFP protein to measure its purity, reagents were made being: 6X DNA gel loading dye (5.6ml of 80% glycerol, 9.2ml MQ water, 0.18ml EDTA at 0.5M and 0.018g bromphenol blue. This wade up to a volume of 15ml) and agarose gel (0.5g DNA grade agarose, 50µL TAE buffer, 2µL cybersafe to 20µL MQ water and 5µL of agarose gel). <br />
The agarose was poured into the plate and cooled. In preparing the samples for the loading gel, 10µL of the YFP samples, 4µL of 6X DNA Loading dye and 10µL of distilled water were added to eppendorf tubes. 12µL of the samples were loaded onto the gel. The plate was put into the tank that was filled with TAE buffer. Electrophoresis was run at 120V for 30 min. <br />
<br />
<br />
|width="50%"|[[Image:SNC00109.jpg|300px|centre|thumb]]<br />
|}<br />
<br><br />
<br />
==Mutagenesis of YFP ==<br />
<br />
'''''Mutant Strand Synthesis Reaction'''''<br />
----<br />
<br />
<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|<br />
The mutagenesis of YFP was completed using Polymerase chain reaction. The control reaction was prepared as indicated below:<br />
<br />
The YFP plasmid was diluted by a 1:17 dilution factor to obtain a concentration of 2.13 µL.<br />
|width="50%"| Lastly, 1µL of DNA polymerase was added. The reaction was done using 18 cycles with the following cycling parameters: <br />
|-<br />
|width="50%"|Primer 657 was diluted to a concentration of 24.1ng/µL using a 1:5 dilution and primer 656 was diluted to 27.1ng/µL using a 1:9 dilution. These dilutions were done to bring the concentrations of the plasmids to 125ng. <br />
Following the dilutions the following was carried out:<br />
<br />
5µL of 10X reaction buffer, 2.13µL YFP plasmid, 5.18µL 657 primer, 4.61µL 656 primer, 1µL of dNTP mix, 3µL of quick solution and 29.08µL of MQ water were added to a eppendorf tube making a total volume of 50µL. <br />
<br />
|width="50%" align="center"|<br />
{| class="wikitable" border="1"<br />
|-<br />
! Segments<br />
! Cycles<br />
! Temperature<br />
! Time<br />
|-<br />
| 1<br />
| 1<br />
| 95°C<br />
| 1 minute<br />
|-<br />
| 2<br />
| 18<br />
| 95°C<br />
| 50 seconds<br />
|-<br />
| <br />
| <br />
| 60°C<br />
| 50 seconds<br />
|-<br />
| <br />
| <br />
| 68°C<br />
| 1 minute<br />
|-<br />
| 3<br />
| 1<br />
| 68°C<br />
| 7 minutes<br />
|}<br />
<br />
|}<br />
<br />
'''''Dpn 1 Digestion of the Amplification Product'''''<br />
----<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|1µL of the Dpn I restriction enyme at 10 U/µL was directly added the amplifictaion reaction in the tube and using a pipette it was thoroguhly mixed by pipetting the solution up and down several times. The reaction mixture was microcentrifuged<br />
|width="50%" | for 1 minute and incubated at 37°C for 7 hours to digest the parental supercoiled dsDNA.</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Project/Materials_and_MethodsTeam:Victoria Australia/Project/Materials and Methods2009-10-20T15:15:34Z<p>Muriel aldunate: </p>
<hr />
<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
<br />
'''''<br />
== <div style="width: 60%; height: 60%; color: #50cad; font-size: 25px; ">Materials & Methods</div> ==<br />
'''''<br />
<br />
__TOC__<br />
==Transformation of fluorescent cells==<br />
{|style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top"<br />
|width="50%" |To begin with, all of the required reagent solutions were prepared. LB Agar plates (protocol 1) were made using 10g tryptone digest of casein, 5g yeast extract, 5g NaCl, 15g agar and then made up to 1L using milliQ water. Ampicillin (100μg/ml) was then prepared by adding 1gram of ampicillin and 9 ml (9gram) milliQ water so that the total volume was 10ml. LB media was made using 10g tryptone, 5g yeast extract, 5g NaCl and milliQ water to make the media up to 1L. <br><br />
<br />
The bacterial transformation of both BFP and GFP was completed by firstly Pre-incubating the LB-agar plates at 37°C for 2 hours prior. The Ca-competent cells were thawed on ice (4ºC). <br />
|width="50%"|Plasmid DNA was added and the tubes stored on ice for 30mins. The tubes were transferred to a rack placed in a preheated water bath (45ºC) and incubated for 30secs. The tubes were then transferred to ice (4ºC) and incubated on ice for 2 min. 800μl of SOC medium was added to each tube and Incubated at 37ºC with gentle shaking (no more that 50rpm) for 45min. 50μl of the transformed cells was transferred onto the LB agar plates each containing ampicillin, and spread the cell suspension thoroughly on the surface using a sterile rod (Streaking). Plates were incubated (agar side up) at 37ºC for ~16h. <br />
Transformation of Vic GFP, BFP, YFP, AZ, blueberry and cherry was done using the same protocol as above. <br />
<br><br />
|}<br />
<br />
==Protein expression==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|Following the transformation, protein expression was carried out by adding 100ml of Lb media into two conical flasks that were previously autoclaved. Using a pipette tip a single colony from the transformed bacteria of GFP and BFP was selected and dropped into conical flask. They were then incubated at 240rpm at 38 ºC for 18 hours.<br />
Using the incubated GFP and BFP they were divided into two falcon tubes each and centrifuged at 3000rpm for 20min. The supernatant was emptied (waste) and the pellet was resuspended with 20ml of SOC solution and 20 µL of ampicillin being added. At a wavelength of 600nm, the solution was measured in the spectrometer to check the density of the bacteria.The results obtained were as follows: GFP = 0.93A and BFP = 0.95A.Two conical flasks were made <br />
|width="50%" |up with 120ml SOC solution and 120 µL of Ampicillin (this is the solution in which GFP and BFP are added to).<br />
Using C1V1=C2V2 the amount of GFP and BFP that was added to the solution from above (SOC and Ampicillin) was found.After adding the GFP/BFP the solution was incubated at 240rpm at 28 ºC for 1 hour. Following this hour the absorbance was read at 600nm and the following results were obtained: GFP = 0.7A (undiluted) and BFP = 0.64A (undiluted).<br />
The proteins were then induced by adding 147µL of IPTG into each conical flask. This was poured into 2 50 ml falcon tubes and centrifuged for 10 min at 2000rpm. Stored at -80°C. <br />
<br />
|}<br />
<br />
==Preparation of reagents==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer. <br />
|width="50%" |100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution. 1M imieazole was prepared by adding 0.6808g of imieazole to 10ml of milliQ water. <br />
|}<br />
==Purification of GFP and BFP==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or BFP. That is; GFP with lysis buffer added to pellet with GFP and BFP with lysis buffer added to pellet with BFP. These were sonicated to split the cells open at 70% duty cycle 30seconds on and 20seconds off for 3 cycles. Samples then centrifuged for 40min. The supernatant was transferred into eppendorf tubes (2 times GFP and 2times BFP). 250 µL of resin was added to each tube and placed on suspension mixer for 20min. again, the supernatant was removed and added 1ml phosphate buffer to the remaining resin. <br />
This was centrifuged at 500rpm for 2min and the supernatant removed with 1ml phosphate buffer being again added. This was centrifuged at 500rpm for 2min and the supernatant removed. The pellet was resuspended with 5ml elution buffer (An elution buffer was made using 2.5ml imieazole with phosphate buffer being added resulting in a total volume of 10ml. ) again this was centrifuged for 5min at 500rpm. The resulting supernatant formed contained the GFP and BFP; this was removed. <br />
<br />
<br />
|width="50%"|<br />
[[Image:SNC00184.jpg|300px|centre|Purified GFP and BFP obtained in the supernatant|thumb]]<br />
|}<br />
<br />
==Preparation of yellow plasmid with E coli bacteria==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|A 6M guanidine hydrochloride solution was prepared along with 50ml Tris Buffer at pH 7.8 ( 1M Tris-HCL with the pH adjusted by adding NaOH). 4M Guanidinium in mix with buffer was added to the protein solution and spun for 30 minutes. This was then centrifuged at 20233rcf and the supernatant put in eppendorf tubes with resin. The ampicillin was then incubated with 1 micro litre/ml working stock 100µg/ml. <br />
4ml of LB medium was put into a flacon tube. With a pipette tip E.coli bacteria was scraped and placed into the falcon tube. It was then incubated overnight at 37 ºC 300rpm. 100ml 2YT solution added to 4 conical flasks. 1ml of the incubated Bactria stock (from above) was put into each flask and incubated at 180rpm 37 ºC. <br />
|width="50%" |After incubation it was centrifuged at 2880rpm for 20min and the supernatant poured into a conical flask (2YT) with decon disinfectant to kill off any remaining bacteria. 8 falcon tubes with the bacteria pellet were then stored (frozen). 6ml of LB media was added to 4 times 15ml falcon tubes. 0.1µl/nl = 6C ampicillin was put in and a stab of yellow fluorescent protein (3.1) was then added to the solution and resuspended. This was incubated at 190rpm at 37 ºC for 16 hours. Following incubation, the solution was centrifuged at 21000rpm for 5 min.<br />
|}<br />
<br />
==Purification of yellow fluorescent plasmid==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|The aurum plasmid spin format protocol was completed (The plasmid containing bacterial host of the yellow protein was transferred to a 1.5-2.0ml capped microcentrifuge tube. Cells were pelleted for 1 min at full speed and the supernatant removed. 250µL of resuspension solution was added and pipetted up and down until the cell pellet was completely resuspended. 250µL of lysis solution was added and mixed by inverting. 5 min after the lysis was added, 350µL of neutralisation solution was added and also mixed by inversion. The neutralised lysate was centrifuged for 5min at 21000rpm {the supernatant or cleared lysate contains the plasmid DNA}.<br />
While centrifuging, a plasmid mini column was inserted into a 2ml capless wash tube. The supernatant was transferred to the plasmid mini column and centrifuged for 1min.<br />
|width="50%" |Wash solution at 5X concentrate was used to add 4 volumes (100ml) of 95-100% ethanol. The plasmid mini column from the wash tube was removed and the filtrate discarded from the tube. The column was replaced into the same wash tube. 750µL of the wash solution was added and centrifuged for 1min. The wash solution was discarded from the tube and the column was replaced into the same wash tube. It was then centrifuged for an additional 1 min to remove the residual wash solution. The plasmid mini column was transferred to a 1.5-2.0ml capped microcentrifuge tube and <br />
50µL of elution solution was added onto the membrane stack at the base of the column and allowed a minute for the solution to saturate the membranes. It was again centrifuged for 1min to elute the plasmid. The mini column was discarded and the eluted DNA was stored in the freezer).<br />
|}<br />
<br />
==Preparation of the E.coli cell free system==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|The E.Coli cell free system was constructed via the following method:<br />
Two buffers s30a and s30b were first made (1.725g potassium acetate, 2.944g magnesium sulphate, added 400ml MQ water and set to a pH of 7.8 by adding trisacetate. It was then filtered in to 2X 250ml beakers one being for S30A in which 500µL of 2 mercaptoethanol was added and S30B that did not contain 2 mercaptoehanol).<br />
The bacteria cells were thawed on ice and resuspended the pellets by washing out with S30A buffer and all he solution transferred into 2X falcon tubes. <br />
|width="50%" |These were then centrifuged at 2000 RCF for 10 minutes. Again, the pellets were resuspended with S30A buffer and centrifuged at 2000 RCF for 10 minutes. This was completed twice as to a total of 3 times. The supernatant was removed. The pellet was resuspended with S30B buffer and then sonicated for 30 sec on and 30 sec off at 50% for a total of 5 minutes. <br />
1ml samples were placed 10 eppendorf tubes and centrifuged at 20 000 for 30 minutes at 4°C. Supernatant was transferred into clean eppendorf tubes and also centrifuged at 20000 rpm for 45 minutes. 7.25ml of the supernatant was removed = our E.coli cell free system. <br />
|}<br />
<br />
==Expression and purification of yellow and green fluorescent protein==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|An expression and purification of the yellow and green fluorescent proteins was carried out to determine what cell line it had; DH5 alpha or BL21DE3.<br />
An overnight culture was set up in 2X falcon tubed with 5ml LB media and a pipette that was stabbed with YFP and GFP. 5µL of ampicillin was added and incubated overnight at 37°C at 220rpm. The protein expression was then completed by dividing the incubated YFP and GFP into 2X falcon tubes and centrifuged for 20 minutes at 3000rpm. <br />
The supernatant was emptied and the pellet resuspended with 20 ml of SOC solution and 20µL of ampicillin added. <br />
Spectrophotemetric analysis of the GFP and YFP at 1:100 dilution was done in the nanodrop 2000. <br />
|width="50%" |Absorbance at 600nm was taken before the dilution and the results were as follows: GFP=1.4 and YFP=0.8. the overnight cultures were then spun in the centrifuge to create a pellet for resuspension and again incubated at 37°C and 220rpm. The OD measurement was taken to be: YFP = 0.195 and GFP = 0.374.<br />
The proteins were then induced by adding 147µL of IPTG into each conical flask containing the YFP and GFP. This was poured into 2 50 ml falcon tubes and centrifuged for 10 min at 2000rpm. Stored at -80°C.<br />
|}<br />
<br />
==Electrophoresis of YFP==<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|In preparation for the electrophoresis run of the YFP protein to measure its purity, reagents were made being: 6X DNA gel loading dye (5.6ml of 80% glycerol, 9.2ml MQ water, 0.18ml EDTA at 0.5M and 0.018g bromphenol blue. This wade up to a volume of 15ml) and agarose gel (0.5g DNA grade agarose, 50µL TAE buffer, 2µL cybersafe to 20µL MQ water and 5µL of agarose gel). <br />
The agarose was poured into the plate and cooled. In preparing the samples for the loading gel, 10µL of the YFP samples, 4µL of 6X DNA Loading dye and 10µL of distilled water were added to eppendorf tubes. 12µL of the samples were loaded onto the gel. The plate was put into the tank that was filled with TAE buffer. Electrophoresis was run at 120V for 30 min. <br />
<br />
<br />
|width="50%"|[[Image:SNC00109.jpg|300px|centre|thumb]]<br />
|}<br />
<br><br />
<br />
==Mutagenesis of YFP ==<br />
<br />
'''''Mutant Strand Synthesis Reaction'''''<br />
----<br />
<br />
<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|<br />
The mutagenesis of YFP was completed using Polymerase chain reaction. The control reaction was prepared as indicated below:<br />
<br />
The YFP plasmid was diluted by a 1:17 dilution factor to obtain a concentration of 2.13 µL.<br />
|width="50%"| Lastly, 1µL of DNA polymerase was added. The reaction was done using 18 cycles with the following cycling parameters: <br />
|-<br />
|width="50%"|Primer 657 was diluted to a concentration of 24.1ng/µL using a 1:5 dilution and primer 656 was diluted to 27.1ng/µL using a 1:9 dilution. These dilutions were done to bring the concentrations of the plasmids to 125ng. <br />
Following the dilutions the following was carried out:<br />
<br />
5µL of 10X reaction buffer, 2.13µL YFP plasmid, 5.18µL 657 primer, 4.61µL 656 primer, 1µL of dNTP mix, 3µL of quick solution and 29.08µL of MQ water were added to a eppendorf tube making a total volume of 50µL. <br />
<br />
|width="50%" align="center"|<br />
{| class="wikitable" border="1"<br />
|-<br />
! Segments<br />
! Cycles<br />
! Temperature<br />
! Time<br />
|-<br />
| 1<br />
| 1<br />
| 95°C<br />
| 1 minute<br />
|-<br />
| 2<br />
| 18<br />
| 95°C<br />
| 50 seconds<br />
|-<br />
| <br />
| <br />
| 60°C<br />
| 50 seconds<br />
|-<br />
| <br />
| <br />
| 68°C<br />
| 1 minute<br />
|-<br />
| 3<br />
| 1<br />
| 68°C<br />
| 7 minutes<br />
|}<br />
<br />
|}<br />
<br />
'''''Dpn 1 Digestion of the Amplification Product'''''<br />
----<br />
{| style="text-align:center"; width="100%"<br />
|- <br />
|-valign="top" <br />
|width="50%"|1µL of the Dpn I restriction enyme at 10 U/µL was directly added the amplifictaion reaction in the tube and using a pipette it was thoroguhly mixed by pipetting the solution up and down several times. The reaction mixture was microcentrifuged<br />
|width="50%" | for 1 minute and incubated at 37°C for 7 hours to digest the parental supercoiled dsDNA.</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Concept/Team_NameTeam:Victoria Australia/Concept/Team Name2009-10-20T15:05:12Z<p>Muriel aldunate: </p>
<hr />
<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
<br />
<br />
<br />
<br />
== TEAM NAME ==<br />
<br />
<html><br />
<body><br />
<br />
<img src="http://www.livepixel.net.au/igemteamname_defined.jpg" alt="Annotated version of team name, logo and mascot"><br />
<br />
</body><br />
</html><br />
<br />
== TEAM MASCOT ==<br />
The Koala in the Lab coat: "'''Fluoro Lou'''"<br />
<br />
Although also short for Louis, our mascot has been called Lou as a play on words for the two letters: '''Lu'''<br />
<br />
These letters form the beginning of the Latin words '''Lu'''men, '''Lu'''x and '''Lu'''z all used as names for Light.<br />
<br />
Lou is a Koala, one of many distinct Australian animals, he sports a lab coat which shows he's ensuring his personal protection in the laboratory whilst creating a fluorescent bioligical lighting system that he has used to decorate the Eucalyptus leaves he is grasping.<br />
<br />
<br />
== ALTERNATIVE TEAM NAME & MASCOT IDEAS ==<br />
- Fluoro Future<br />
<br />
- Ecoluminocity<br />
<br />
- The E.colighting Project<br />
<BR><br />
___________________________________________<br />
<br />
- A Fluorescent Robot<br />
<br />
- A Fluorescent Beaver<br />
<br />
- A Fluorescent Tasmanian Devil</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Team/OverviewTeam:Victoria Australia/Team/Overview2009-10-20T15:01:48Z<p>Muriel aldunate: </p>
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{{:Team:Victoria_Australia/Menu}}<br />
{{:Team:Victoria_Australia/CSS}}<br />
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<!--- The Mission, Experiments ---><br />
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== '''<div style="width: 100%; height: 100%; color: #120cad; font-size: 25px; ">Who We Are:</div>''' ==<br />
{|border = "0"<br />
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[[Image:rmit_team_and_lou.jpg|center|frame|Team Victoria]]<br />
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<br />
'''<div style="width: 100%; height: 100%; color: #80f069; font-size: 20px;">ADVISORS:</div>'''<br />
<br />
<br />
*''' Professor Philip Poronnik ''':<br />
<br />
<br />
<br />
[[Image:Phil.P Wiki.jpg|right|frame]]<br />
Professor Philip Poronnik initiated RMIT University iGEM participation and is the overall iGEM leader for Team Victoria_Australia. Prof. Phil participated in iGEM 2008 where he also became known as "P2" in iGEM circles with "P1" being Prof. Phillip Long of UQ/MIT fame. Prof Phil - P2 - is the Discipline Head of Pharmaceutical Sciences at RMIT University and is leader of the Health Innovations Research Institute (HIRi) program in Metabolism, Exercise and Disease. <br />
<br />
Prof. Phil’s research spans areas of molecular physiology of membrane transport; kidney physiology and albumin handling; the physiology, function and regulation of ion channels; the development of innovative teaching methods; and synthetic biology. Prof. Phil has research grants from both the Australian NHMRC and ARC funding bodies and has over 90 publications. <br />
<br />
In addition to his scientific research, Professor Phil is heavily involved in science education research. He is an Associate Fellow of the Australian Learning and Teaching Council and Research Professorial<br />
Fellow at the Centre for Educational Innovation and Technology at the University of Queensland, directed by P1. His key focus in teaching are areas around creativity and innovation and embedding transferable skills to science graduates - opportunities provided by activities such as iGEM.<br />
<br />
Prof Phil has many recognitions of his outstanding achievements including: <br />
<br>- A UQ Foundation Research Excellence Award (2004). <br />
<br>- “In Focus” review editor for the International Journal of Biochemistry and Cell Biology, <br />
<br>- Editorial Board for the American Journal of Physiology Renal Reviews.<br />
<br>- National Secretary of the Australian and New Zealand Association for the Advancement of Science<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
*'''Dr Leonard Pattenden ''':<br />
[[Image:Len Wiki.jpg|right|frame]]<br />
<br />
<br />
<br />
Dr Len Pattenden is a Senior Postdoctoral Fellow in the Poronnik group of the Health Innovations Research Institute, RMIT University. Len is the RMIT University iGEM team coordinator. Len undertakes research into albumin handling by the kidneys and the relationship of albumin structure to changes in the way it is processed by the kidneys. This research is relevant to diabetes where albumin is modified by sugars and for ~16,000 Australians progress to the complication of albuminuria, which in turn progresses to renal failure and cardiovascular disease. Len has research interests in areas of Synchrotron science, membrane biology, large-scale protein expression, protein engineering, biomolecular engineering, molecular physiology, structural biology, drug and vaccine design, and of course, synthetic biology.<br />
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'''<div style="width: 100%; height: 100%; color: #80f069; font-size: 20px;">UNDERGRADS:</div>'''<br />
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<br />
[[Image:sebastianramov.jpg|right|frame]]<br />
<br />
<br />
*'''Sebastian Ramov''': <br />
Sebastian is in his second year second year of Bachelor Applied science (Pharmaceutical science). He joined the iGEM competition to enhance his knowledge in the lab and to get a feel of what it's like to be a research scientist.<br />
<br />
<br />
<br />
<br />
<br />
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<br />
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<br />
<br />
[[Image:michellechayeb3.jpg|right|frame]]<br />
<br />
<br />
<br />
<br />
*'''Michelle Chayeb''':<br />
Michelle Chayeb is a 20 year old pharmaceutical sciences student and is currently in her second year. Her primary passion lies in drug development and enjoys working in the lab. <br />
<br />
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<br />
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[[Image:lynathach.jpg|right|frame]]<br />
<br />
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*'''Lyna Thach''':<br />
Lyna is currently studying an undergraduate degree at RMIT University; Bachelor of Applied Science (Pharmaceutical Sciences). She enjoys undertaking hands on work that involve chemicals, drugs and skilled work activities. By all means they’ve already got her best interest at heart. She decided to be part of the iGEM competition to broaden her knowledge in the laboratory field, and to have a feel of what it would be like to undertake some of the responsibilities of an Honours student. <br />
<br />
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[[Image:danniikamoto.jpg|right|frame]]<br />
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*'''Danielle Kamato''':<br />
Danielle Kamato is 19 years old and currently studying second year Pharmaceutical Science at RMIT university. Dannii decided to join iGEM so she would have an opportunity to research and develop a project of personal interest, as well as experience lab work first hand.<br />
<br />
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<br />
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[[Image:Jazz.jpg|right|frame]]<br />
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*'''Jasmine Panthaki''':<br />
Jasmine is currently studying her first year of her undergraduate degree at RMIT University; Bachelor of Applied Science (Pharmaceutical Sciences). Her love of science developed in primary school, learning simple things about the body and space. She is determined to gain as much knowledge as she can about the mechanism of drugs and the immune system and after completing her degree she plans to travel throughout Africa.<br />
<br />
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<br />
[[Image:Science.jpg|right|frame]]<br />
<br />
<br />
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*'''Flora Yuen''':<br />
Flora is currently studying her first year of her undergraduate degree at RMIT University; Bachelor of Applied Science (Pharmaceutical Sciences). She has a huge passion in the field of science, although she can complain about the workload and information overload alot, deep down she loves it. She hopes that in the future that science will take her around the world. <br />
iGEM was brought to her attention since it would be a great opportunity to explore an aspect of science in great detail, while having fun.<br />
<br />
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[[Image:monkeymu.jpg|right|frame]]<br />
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*'''Muriel Aldunate''':<br />
Muriel is in her 2nd year of studying biotechnology at RMIT University and has dedicated her life to science since she was five; when her parents bought her a microscope and she went around collecting helpless insects to inspect. In her spare time she aims to inspire future generations by assisting by teaching year 7 science & being a rolemodel that can turn "geek into chic". She plans never to stop travelling the world, marvelling at its wonders and trying to understand everything in it.<br />
<br />
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<br />
[[Image:Halloween.jpg|right|frame]]<br />
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*'''Candice Raeburn''': <br />
Candi is a 2nd year biotechnology student who enjoys making surprised facial expressions at beer in conical flasks (see picture right) and genetics. When she grows up she hopes to be taller and intends to save the world (to fulfil the young optimistic scientist stereotype). Candi enjoys: bioacoustics, behavioural genetics, entomology, biological chemistry, virology, biomimicry, nanotechnology and a nice pair of slacks. She has studied in Australia and the USA, and hopes to complete her degree in the UK next year.<br />
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[[Image:breegini.jpg|right|frame]]<br />
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*'''Breegini Culas''':<br />
Breegini is a 1st year student studying an undergraduate degree in Applied Science (Pharmaceutical Science) at RMIT. Science has always been an interest in her life, she loves doing hands on lab work more than anything. iGEM sparked her interest because she wanted a challenge and this gave rise to a great opportunity to travel as well.</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_AustraliaTeam:Victoria Australia2009-10-20T15:00:36Z<p>Muriel aldunate: </p>
<hr />
<div>'''''<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
{{:Team:Victoria_Australia/CSS}}<br />
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== <div style="width: 60%; height: 60%; color: #50cad; font-size: 25px; ">Preliminary Project Outline</div> ==<br />
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The RMIT University iGEM (Victoria_Australia) team aims to build a biological lighting system via cell free transcription and translation also known as CFTnT technology. This project aims to apply the principles of Synthetic Biology in a simple manner that could be efficient for everyday use. The project concept was developed after the consideration of current energy and climate change issues. <br />
A CFTnT system was chosen because in principle, the lysate used in the CFTnT system may be prepared from any type of cell. Hence, the team thought it would be of merit to develop an alternative light source, which could possibly be powered by a waste material as simple as grass clippings used as the cell lysate. The team also hopes to address cellular and bioprocess problems that arise when employing a CFTnT system and is considering many novel approaches to creating a more sustainable system that can be scaled up for practical applications. Initial experiments conducted by Victoria_Australia RMIT University team will focus on two cell-free systems derived from ''Escherichia coli'' and wheat germ. <br />
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<span style="font-weight: bold;">Current Status:</span> With lab work nearing to an end, we have created an E.Coli cell free system, transformed and purified fluorescent proteins and are currently working on expressing the proteins in the cell free system and making an iGEM biobrick. Many hours are being put in to finish our presentation and poster with the jamboree not too far away. <br />
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{{:Team:Victoria_Australia/countdown|center}}</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Team/OverviewTeam:Victoria Australia/Team/Overview2009-10-20T14:53:18Z<p>Muriel aldunate: </p>
<hr />
<div>[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
{{:Team:Victoria_Australia/CSS}}<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
<br />
== '''<div style="width: 100%; height: 100%; color: #120cad; font-size: 25px; ">Who We Are</div>''' ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
<br />
[[Image:rmit_team_and_lou.jpg|center|frame|Team Victoria]]<br />
<br />
<br />
<br />
'''<div style="width: 100%; height: 100%; color: #80f069; font-size: 20px;">ADVISORS:</div>'''<br />
<br />
<br />
*''' Professor Philip Poronnik ''':<br />
<br />
<br />
<br />
[[Image:Phil.P Wiki.jpg|right|frame]]<br />
Professor Philip Poronnik initiated RMIT University iGEM participation and is the overall iGEM leader for Team Victoria_Australia. Prof. Phil participated in iGEM 2008 where he also became known as "P2" in iGEM circles with "P1" being Prof. Phillip Long of UQ/MIT fame. Prof Phil - P2 - is the Discipline Head of Pharmaceutical Sciences at RMIT University and is leader of the Health Innovations Research Institute (HIRi) program in Metabolism, Exercise and Disease. <br />
<br />
Prof. Phil’s research spans areas of molecular physiology of membrane transport; kidney physiology and albumin handling; the physiology, function and regulation of ion channels; the development of innovative teaching methods; and synthetic biology. Prof. Phil has research grants from both the Australian NHMRC and ARC funding bodies and has over 90 publications. <br />
<br />
In addition to his scientific research, Professor Phil is heavily involved in science education research. He is an Associate Fellow of the Australian Learning and Teaching Council and Research Professorial<br />
Fellow at the Centre for Educational Innovation and Technology at the University of Queensland, directed by P1. His key focus in teaching are areas around creativity and innovation and embedding transferable skills to science graduates - opportunities provided by activities such as iGEM.<br />
<br />
Prof Phil has many recognitions of his outstanding achievements including: <br />
<br>- A UQ Foundation Research Excellence Award (2004). <br />
<br>- “In Focus” review editor for the International Journal of Biochemistry and Cell Biology, <br />
<br>- Editorial Board for the American Journal of Physiology Renal Reviews.<br />
<br>- National Secretary of the Australian and New Zealand Association for the Advancement of Science<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
*'''Dr Leonard Pattenden ''':<br />
[[Image:Len Wiki.jpg|right|frame]]<br />
<br />
<br />
<br />
Dr Len Pattenden is a Senior Postdoctoral Fellow in the Poronnik group of the Health Innovations Research Institute, RMIT University. Len is the RMIT University iGEM team coordinator. Len undertakes research into albumin handling by the kidneys and the relationship of albumin structure to changes in the way it is processed by the kidneys. This research is relevant to diabetes where albumin is modified by sugars and for ~16,000 Australians progress to the complication of albuminuria, which in turn progresses to renal failure and cardiovascular disease. Len has research interests in areas of Synchrotron science, membrane biology, large-scale protein expression, protein engineering, biomolecular engineering, molecular physiology, structural biology, drug and vaccine design, and of course, synthetic biology.<br />
<br />
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'''<div style="width: 100%; height: 100%; color: #80f069; font-size: 20px;">UNDERGRADS:</div>'''<br />
<br />
<br />
<br />
<br />
<br />
[[Image:sebastianramov.jpg|right|frame]]<br />
<br />
<br />
*'''Sebastian Ramov''': <br />
Sebastian is in his second year second year of Bachelor Applied science (Pharmaceutical science). He joined the iGEM competition to enhance his knowledge in the lab and to get a feel of what it's like to be a research scientist.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:michellechayeb3.jpg|right|frame]]<br />
<br />
<br />
<br />
<br />
*'''Michelle Chayeb''':<br />
Michelle Chayeb is a 20 year old pharmaceutical sciences student and is currently in her second year. Her primary passion lies in drug development and enjoys working in the lab. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:lynathach.jpg|right|frame]]<br />
<br />
<br />
<br />
*'''Lyna Thach''':<br />
Lyna is currently studying an undergraduate degree at RMIT University; Bachelor of Applied Science (Pharmaceutical Sciences). She enjoys undertaking hands on work that involve chemicals, drugs and skilled work activities. By all means they’ve already got her best interest at heart. She decided to be part of the iGEM competition to broaden her knowledge in the laboratory field, and to have a feel of what it would be like to undertake some of the responsibilities of an Honours student. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:danniikamoto.jpg|right|frame]]<br />
<br />
<br />
<br />
<br />
<br />
*'''Danielle Kamato''':<br />
Danielle Kamato is 19 years old and currently studying second year Pharmaceutical Science at RMIT university. Dannii decided to join iGEM so she would have an opportunity to research and develop a project of personal interest, as well as experience lab work first hand.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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<br />
<br />
<br />
[[Image:Jazz.jpg|right|frame]]<br />
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<br />
<br />
<br />
<br />
*'''Jasmine Panthaki''':<br />
Jasmine is currently studying her first year of her undergraduate degree at RMIT University; Bachelor of Applied Science (Pharmaceutical Sciences). Her love of science developed in primary school, learning simple things about the body and space. She is determined to gain as much knowledge as she can about the mechanism of drugs and the immune system and after completing her degree she plans to travel throughout Africa.<br />
<br />
<br />
<br />
<br />
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<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:Science.jpg|right|frame]]<br />
<br />
<br />
<br />
*'''Flora Yuen''':<br />
Flora is currently studying her first year of her undergraduate degree at RMIT University; Bachelor of Applied Science (Pharmaceutical Sciences). She has a huge passion in the field of science, although she can complain about the workload and information overload alot, deep down she loves it. She hopes that in the future that science will take her around the world. <br />
iGEM was brought to her attention since it would be a great opportunity to explore an aspect of science in great detail, while having fun.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:monkeymu.jpg|right|frame]]<br />
<br />
<br />
<br />
*'''Muriel Aldunate''':<br />
Muriel is in her 2nd year of studying biotechnology at RMIT University and has dedicated her life to science since she was five; when her parents bought her a microscope and she went around collecting helpless insects to inspect. In her spare time she aims to inspire future generations by assisting by teaching year 7 science & being a rolemodel that can turn "geek into chic". She plans never to stop travelling the world, marvelling at its wonders and trying to understand everything in it.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:Halloween.jpg|right|frame]]<br />
<br />
<br />
<br />
*'''Candice Raeburn''': <br />
Candi is a 2nd year biotechnology student who enjoys making surprised facial expressions at beer in conical flasks (see picture right) and genetics. When she grows up she hopes to be taller and intends to save the world (to fulfil the young optimistic scientist stereotype). Candi enjoys: bioacoustics, behavioural genetics, entomology, biological chemistry, virology, biomimicry, nanotechnology and a nice pair of slacks. She has studied in Australia and the USA, and hopes to complete her degree in the UK next year.<br />
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<br />
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<br />
[[Image:breegini.jpg|right|frame]]<br />
<br />
<br />
<br />
*'''Breegini Culas''':<br />
Breegini is a 1st year student studying an undergraduate degree in Applied Science (Pharmaceutical Science) at RMIT. Science has always been an interest in her life, she loves doing hands on lab work more than anything. iGEM sparked her interest because she wanted a challenge and this gave rise to a great opportunity to travel as well.</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Team/OverviewTeam:Victoria Australia/Team/Overview2009-10-20T14:41:32Z<p>Muriel aldunate: </p>
<hr />
<div>[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
{{:Team:Victoria_Australia/CSS}}<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
<br />
== '''<div style="width: 100%; height: 100%; color: #120cad; font-size: 25px; ">Who We Are</div>''' ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
<br />
[[Image:rmit_team_and_lou.jpg|center|frame|Team Victoria]]<br />
<br />
<br />
<br />
'''<div style="width: 100%; height: 100%; color: #80f069; font-size: 20px;">ADVISORS:</div>'''<br />
<br />
<br />
*''' Professor Philip Poronnik ''':<br />
<br />
<br />
<br />
[[Image:Phil.P Wiki.jpg|right|frame]]<br />
Professor Philip Poronnik initiated RMIT University iGEM participation and is the overall iGEM leader for Team Victoria_Australia. Prof. Phil participated in iGEM 2008 where he also became known as "P2" in iGEM circles with "P1" being Prof. Phillip Long of UQ/MIT fame. Prof Phil - P2 - is the Discipline Head of Pharmaceutical Sciences at RMIT University and is leader of the Health Innovations Research Institute (HIRi) program in Metabolism, Exercise and Disease. <br />
<br />
Prof. Phil’s research spans areas of molecular physiology of membrane transport; kidney physiology and albumin handling; the physiology, function and regulation of ion channels; the development of innovative teaching methods; and synthetic biology. Prof. Phil has research grants from both the Australian NHMRC and ARC funding bodies and has over 90 publications. <br />
<br />
In addition to his scientific research, Professor Phil is heavily involved in science education research. He is an Associate Fellow of the Australian Learning and Teaching Council and Research Professorial<br />
Fellow at the Centre for Educational Innovation and Technology at the University of Queensland, directed by P1. His key focus in teaching are areas around creativity and innovation and embedding transferable skills to science graduates - opportunities provided by activities such as iGEM.<br />
<br />
Prof Phil has many recognitions of his outstanding achievements including: <br />
<br>- A UQ Foundation Research Excellence Award (2004). <br />
<br>- “In Focus” review editor for the International Journal of Biochemistry and Cell Biology, <br />
<br>- Editorial Board for the American Journal of Physiology Renal Reviews.<br />
<br>- National Secretary of the Australian and New Zealand Association for the Advancement of Science<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
*'''Dr Leonard Pattenden ''':<br />
[[Image:Len Wiki.jpg|right|frame]]<br />
<br />
<br />
<br />
Dr Len Pattenden is a Senior Postdoctoral Fellow in the Poronnik group of the Health Innovations Research Institute, RMIT University. Len is the RMIT University iGEM team coordinator. Len undertakes research into albumin handling by the kidneys and the relationship of albumin structure to changes in the way it is processed by the kidneys. This research is relevant to diabetes where albumin is modified by sugars and for ~16,000 Australians progress to the complication of albuminuria, which in turn progresses to renal failure and cardiovascular disease. Len has research interests in areas of Synchrotron science, membrane biology, large-scale protein expression, protein engineering, biomolecular engineering, molecular physiology, structural biology, drug and vaccine design, and of course, synthetic biology.<br />
<br />
<br><br />
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<br />
'''<div style="width: 100%; height: 100%; color: #80f069; font-size: 20px;">UNDERGRADS:</div>'''<br />
<br />
<br />
<br />
<br />
*'''Sebastian Ramov''':<br />
[[Image:sebastianramov.jpg|right|frame]]<br />
Sebastian is in his second year second year of Bachelor Applied science (Pharmaceutical science). He joined the iGEM competition to enhance his knowledge in the lab and to get a feel of what it's like to be a research scientist.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
*'''Michelle Chayeb''':<br />
[[Image:michellechayeb3.jpg|right|frame]]<br />
Michelle Chayeb is a 20 year old pharmaceutical sciences student and is currently in her second year. Her primary passion lies in drug development and enjoys working in the lab. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
*'''Lyna Thach''':<br />
[[Image:lynathach.jpg|right|frame]]<br />
Lyna is currently studying an undergraduate degree at RMIT University; Bachelor of Applied Science (Pharmaceutical Sciences). She enjoys undertaking hands on work that involve chemicals, drugs and skilled work activities. By all means they’ve already got her best interest at heart. She decided to be part of the iGEM competition to broaden her knowledge in the laboratory field, and to have a feel of what it would be like to undertake some of the responsibilities of an Honours student. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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*'''Danielle Kamato''':<br />
[[Image:danniikamoto.jpg|right|frame]]<br />
Danielle Kamato is 19 years old and currently studying second year Pharmaceutical Science at RMIT university. Dannii decided to join iGEM so she would have an opportunity to research and develop a project of personal interest, as well as experience lab work first hand.<br />
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*'''Jasmine Panthaki''':<br />
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Jasmine is currently studying her first year of her undergraduate degree at RMIT University; Bachelor of Applied Science (Pharmaceutical Sciences). Her love of science developed in primary school, learning simple things about the body and space. She is determined to gain as much knowledge as she can about the mechanism of drugs and the immune system and after completing her degree she plans to travel throughout Africa.<br />
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*'''Flora Yuen''':<br />
[[Image:Science.jpg|right|frame]]<br />
Flora is currently studying her first year of her undergraduate degree at RMIT University; Bachelor of Applied Science (Pharmaceutical Sciences). She has a huge passion in the field of science, although she can complain about the workload and information overload alot, deep down she loves it. She hopes that in the future that science will take her around the world. <br />
iGEM was brought to her attention since it would be a great opportunity to explore an aspect of science in great detail, while having fun.<br />
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*'''Muriel Aldunate''':<br />
[[Image:monkeymu.jpg|right|frame]]<br />
Muriel is in her 2nd year of studying biotechnology at RMIT University and has dedicated her life to science since she was five; when her parents bought her a microscope and she went around collecting helpless insects to inspect. In her spare time she aims to inspire future generations by assisting by teaching year 7 science & being a rolemodel that can turn "geek into chic". She plans never to stop travelling the world, marvelling at its wonders and trying to understand everything in it.<br />
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*'''Candice Raeburn''': <br />
[[Image:Halloween.jpg|right|frame]]<br />
Candi is a 2nd year biotechnology student who enjoys making surprised facial expressions at beer in conical flasks (see picture right) and genetics. When she grows up she hopes to be taller and intends to save the world (to fulfil the young optimistic scientist stereotype). Candi enjoys: bioacoustics, behavioural genetics, entomology, biological chemistry, virology, biomimicry, nanotechnology and a nice pair of slacks. She has studied in Australia and the USA, and hopes to complete her degree in the UK next year.<br />
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*'''Breegini Culas''':<br />
[[Image:breegini.jpg|right|frame]]<br />
Breegini is a 1st year student studying an undergraduate degree in Applied Science (Pharmaceutical Science) at RMIT. Science has always been an interest in her life, she loves doing hands on lab work more than anything. iGEM sparked her interest because she wanted a challenge and this gave rise to a great opportunity to travel as well.</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_AustraliaTeam:Victoria Australia2009-10-20T14:34:59Z<p>Muriel aldunate: </p>
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<div>'''''<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
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== <div style="width: 60%; height: 60%; color: #50cad; font-size: 25px; ">Preliminary Project Outline</div> ==<br />
'''''<br />
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The RMIT University iGEM (Victoria_Australia) team aims to build a biological lighting system via cell free transcription and translation also known as CFTnT technology. This project aims to apply the principles of Synthetic Biology in a simple manner that could be efficient for everyday use. The project concept was developed after the consideration of current energy and climate change issues. <br />
A CFTnT system was chosen because in principle, the lysate used in the CFTnT system may be prepared from any type of cell. Hence, the team thought it would be of merit to develop an alternative light source, which could possibly be powered by a waste material as simple as grass clippings used as the cell lysate. The team also hopes to address cellular and bioprocess problems that arise when employing a CFTnT system and is considering many novel approaches to creating a more sustainable system that can be scaled up for practical applications. Initial experiments conducted by Victoria_Australia RMIT University team will focus on two cell-free systems derived from ''Escherichia coli'' and wheat germ. <br />
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<span style="font-weight: bold;">Current Status:</span> With lab work nearing to an end, we have created an E.Coli cell free system, transformed and purified fluorescent proteins and are currently working on expressing the proteins in the cell free system and making an iGEM biobrick. Many hours are being put in to finish our presentation and poster with the jamboree not too far away. <br />
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{{:Team:Victoria_Australia/countdown|center}}</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/SponsorsTeam:Victoria Australia/Sponsors2009-10-20T14:13:57Z<p>Muriel aldunate: </p>
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<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
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== We Would Like To Thank Our Generous Sponsors==<br />
<br />
{| class="wikitable" border="0" cellpadding="10"<br />
|-<br />
[[Image:RMIT.jpg|400px|thumb|RMIT University, Melbourne, Australia|left]] || The Royal Melbourne Institute of Technology is one of Australia’s original and leading educational institutions with an international reputation for excellence in work-relevant education and high quality research.<br />
|}<br />
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{| class="wikitable" cellpadding="10"<br />
| [[Image:HIRi.gif|thumb|220px|RMIT Health Innovations Research Institute (HIRi), Melbourne|centre]] || RMIT HIRi takes an integrated approach to address key health issues facing the Australian community in the 21st century through research programs focussing on understanding how the human body functions at a molecular and cellular level. This knowledge will inform innovative therapeutic strategies to target disease states.<br />
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| [[Image:csiro.jpg|thumb|200px|CSIRO|centre]] || The Commonwealth Scientific and Industrial Research Organisation is Australia's national science agency and one of the largest and most diverse research agencies in the world.<br />
|-<br />
| [[Image:SEH.gif|thumb|220px|RMIT College of Science, Engineering and Health, Melbourne|centre]] || The RMIT College of Science, Engineering and Health comprises of ten schools across two campuses offering programs that range from certificates through to PhDs.<br />
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|-<br />
| [[Image:qiagen.gif|thumb|Qiagen|centre]] || QIAGEN is the leading provider of sample and assay technologies. Sample technologies are used to isolate DNA, RNA, and proteins from any biological sample. Assay technologies are then used to make specific target biomolecules, such as the DNA of a specific virus, visible for subsequent analysis.<br />
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| '''ACKNOWLEDGMENTS:''' || <br />
We would also like to thank all individuals who contributed to the success of the RMIT University Fluoroforce team:<br />
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-Friends, family and teachers who provided moral support and guidance to the teams' Undergradtuate students<br />
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-Mr Kieran Wilson: Web Content Coordinator for the Science, Engineering & Health Office (RMIT University)<br />
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-Mr Robin Dick: Manager, Benefit Attraction. Alumni and Development (RMIT University)<br />
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-Bennetts Lane Jazz, Farm pro, Journal Cafe and Journal Canteen for their donations</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/SponsorsTeam:Victoria Australia/Sponsors2009-10-20T12:12:17Z<p>Muriel aldunate: </p>
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<div>{{:Team:Victoria_Australia/CSS}}<br />
[[Image:Finalised fluoroforce header.jpg|center]]<br />
{{:Team:Victoria_Australia/Menu}}<br />
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== We Would Like To Thank Our Generous Sponsors==<br />
<br />
{| class="wikitable" border="0" cellpadding="10"<br />
|-<br />
[[Image:RMIT.jpg|400px|thumb|RMIT University, Melbourne, Australia|left]] || The Royal Melbourne Institute of Technology is one of Australia’s original and leading educational institutions with an international reputation for excellence in work-relevant education and high quality research.<br />
|}<br />
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{| class="wikitable" cellpadding="10"<br />
| [[Image:HIRi.gif|thumb|220px|RMIT Health Innovations Research Institute (HIRi), Melbourne|centre]] || RMIT HIRi takes an integrated approach to address key health issues facing the Australian community in the 21st century through research programs focussing on understanding how the human body functions at a molecular and cellular level. This knowledge will inform innovative therapeutic strategies to target disease states.<br />
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|-<br />
| [[Image:csiro.jpg|thumb|200px|CSIRO|centre]] || The Commonwealth Scientific and Industrial Research Organisation is Australia's national science agency and one of the largest and most diverse research agencies in the world.<br />
|-<br />
| [[Image:SEH.gif|thumb|220px|RMIT College of Science, Engineering and Health, Melbourne|centre]] || The RMIT College of Science, Engineering and Health comprises of ten schools across two campuses offering programs that range from certificates through to PhDs.<br />
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|-<br />
| [[Image:qiagen.gif|thumb|Qiagen|centre]] || QIAGEN is the leading provider of sample and assay technologies. Sample technologies are used to isolate DNA, RNA, and proteins from any biological sample. Assay technologies are then used to make specific target biomolecules, such as the DNA of a specific virus, visible for subsequent analysis.<br />
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| '''ACKNOWLEDGMENTS:''' || <br />
We would also like to thank all individuals who contributed non financially to the success of the RMIT University Fluoroforce team:<br />
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-Friends, family and teachers who provided moral support and guidance to the teams' Undergradtuate students<br />
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-Mr Kieran Wilson: Web Content Coordinator for the Science, Engineering & Health Office (RMIT University)<br />
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-Bennetts Lane Jazz, Journal Cafe and Journal Canteen for their donations</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Concept/Team_NameTeam:Victoria Australia/Concept/Team Name2009-10-20T11:42:34Z<p>Muriel aldunate: </p>
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== TEAM NAME ==<br />
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== TEAM MASCOT ==<br />
The Koala in the Lab coat: "'''Fluoro Lou'''"<br />
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Although also short for Louis, our mascot has been called Lou as a play on words for the two letters: '''Lu'''<br />
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These letters form beginning of the Latin words '''Lu'''men, '''Lu'''x and '''Lu'''z all used as names for Light.<br />
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Lou is a Koala, one of many distinct Australian animals, he sports a lab coat which shows he's ensuring his personal protection in the laboratory whilst creating a fluorescent bioligical lighting system that he has used to decorate the Eucalyptus leaves he is grasping.<br />
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== ALTERNATIVE TEAM NAME & MASCOT IDEAS ==<br />
- Fluoro Future<br />
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- Ecoluminocity<br />
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- The E.colighting Project<br />
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- A Fluorescent Robot<br />
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- A Fluorescent Beaver<br />
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- A Fluorescent Tasmanian Devil</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Concept/Team_NameTeam:Victoria Australia/Concept/Team Name2009-10-20T11:41:22Z<p>Muriel aldunate: </p>
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[[Image:Finalised fluoroforce header.jpg|center]]<br />
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== TEAM NAME ==<br />
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== TEAM MASCOT ==<br />
The Koala in the Lab coat: "'''Fluoro Lou'''"<br />
<br />
Although also short for Louis, our mascot has been called Lou as a play on words for the two letters: '''Lu'''<br />
<br />
These letters form beginning of the Latin words '''Lu'''men, '''Lu'''x and '''Lu'''z all used as names for Light.<br />
<br />
Lou is a Koala, one of many distinct Australian animals, he sports a lab coat which shows he's ensuring his personal protection in the laboratory whilst creating a fluorescent bioligical lighting system that he has used to decorate the Eucalyptus leaves he is grasping.<br />
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== OTHER IDEAS ==<br />
- Fluoro Future<br />
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- Ecoluminocity<br />
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- The E.colighting Project<br />
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- A Fluorescent Tasmanian Devil</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Concept/Team_NameTeam:Victoria Australia/Concept/Team Name2009-10-20T11:18:03Z<p>Muriel aldunate: /* FINAL TEAM NAME & MASCOT */</p>
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== FINAL TEAM NAME & MASCOT ==<br />
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== OTHER IDEAS ==<br />
- Fluoro Future<br />
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- Ecoluminocity<br />
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- The E.colighting Project<br />
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___________________________________________<br />
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- A Fluorescent Robot<br />
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- A Fluorescent Beaver<br />
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- A Fluorescent Tasmanian Devil</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Concept/Team_NameTeam:Victoria Australia/Concept/Team Name2009-10-20T11:14:23Z<p>Muriel aldunate: /* FINAL TEAM NAME & MASCOT */</p>
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== FINAL TEAM NAME & MASCOT ==<br />
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== OTHER IDEAS ==<br />
- Fluoro Future<br />
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- Ecoluminocity<br />
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- The E.colighting Project<br />
<BR><br />
___________________________________________<br />
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- A Fluorescent Robot<br />
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- A Fluorescent Beaver<br />
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- A Fluorescent Tasmanian Devil</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Concept/Team_NameTeam:Victoria Australia/Concept/Team Name2009-10-20T10:42:38Z<p>Muriel aldunate: </p>
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- Fluoro Future<br />
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- The E.colighting Project<br />
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___________________________________________<br />
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- A Fluorescent Robot<br />
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- A Fluorescent Beaver<br />
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- A Fluorescent Tasmanian Devil</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Concept/Team_NameTeam:Victoria Australia/Concept/Team Name2009-10-20T10:33:44Z<p>Muriel aldunate: </p>
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== OTHER IDEAS ==<br />
- Fluoro Future<br />
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- Ecoluminocity<br />
<br />
- The E.colighting Project<br />
<BR><br />
___________________________________________<br />
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- A Fluorescent Robot<br />
<br />
- A Fluorescent Beaver<br />
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- A Fluorescent Tasmanian Devil</div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Concept/Team_NameTeam:Victoria Australia/Concept/Team Name2009-10-20T10:23:04Z<p>Muriel aldunate: </p>
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== OTHER IDEAS ==<br />
- Fluoro Future<br />
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- Ecoluminocity<br />
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- The E.colighting Project<br />
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___________________________________________<br />
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- A Fluorescent Robot<br />
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- A Fluorescent Beaver<br />
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- A Fluorescent Tasmanian Devil</div>Muriel aldunatehttp://2009.igem.org/File:Muriel_igemlogo_defined.jpgFile:Muriel igemlogo defined.jpg2009-10-20T09:41:45Z<p>Muriel aldunate: </p>
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<div></div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Concept/The_LogoTeam:Victoria Australia/Concept/The Logo2009-10-20T09:41:04Z<p>Muriel aldunate: </p>
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<a href="https://2009.igem.org/Team:Victoria_Australia" style="color: black">Home<br />
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<li><a href="https://2009.igem.org/Team:Victoria_Australia/Team/Overview"><span><span>Overview</span></span></a></li><br />
<li><a href="https://2009.igem.org/Team:Victoria_Australia/Team/Members"><span><span>Team member profiles</span></span></a></li><br />
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<li><a href="https://2009.igem.org/Team:Victoria_Australia/Concept/Project"><span><span>Project</span></span></a></li><br />
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href="https://2009.igem.org/Team:Victoria_Australia/Concept/Team_Name"><span><span>Team Name & Mascot</span></span></a></li><br />
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<li><a href="https://2009.igem.org/Team:Victoria_Australia/Project/Introduction"><span><span>Introduction</span></span></a></li><br />
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<li><a href="https://2009.igem.org/Team:Victoria_Australia/Considerations#Ethics"><span><span>Ethics</span></span></a></li><br />
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<li><a href="https://2009.igem.org/Team:Victoria_Australia/Team/Overview"><span><span>Overview</span></span></a></li><br />
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</html></div>Muriel aldunatehttp://2009.igem.org/Team:Victoria_Australia/Team/MembersTeam:Victoria Australia/Team/Members2009-09-29T06:44:06Z<p>Muriel aldunate: New page: center {{:Team:Victoria_Australia/Menu}} {{:Team:Victoria_Australia/CSS}} <!--- The Mission, Experiments ---> == '''<div style="width: 100%; h...</p>
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== '''<div style="width: 100%; height: 100%; color: #120cad; font-size: 25px; ">Who We Are</div>''' ==<br />
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'''<div style="width: 100%; height: 100%; color: #80f069; font-size: 20px;">ADVISORS:</div>'''<br />
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*''' Professor Philip Poronnik ''':<br />
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Professor Philip Poronnik initiated RMIT University iGEM participation and is the overall iGEM leader for Team Victoria_Australia. Prof. Phil participated in iGEM 2008 where he also became known as "P2" in iGEM circles with "P1" being Prof. Phillip Long of UQ/MIT fame. Prof Phil - P2 - is the Discipline Head of Pharmaceutical Sciences at RMIT University and is leader of the Health Innovations Research Institute (HIRi) program in Metabolism, Exercise and Disease. <br />
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Prof. Phil’s research spans areas of molecular physiology of membrane transport; kidney physiology and albumin handling; the physiology, function and regulation of ion channels; the development of innovative teaching methods; and synthetic biology. Prof. Phil has research grants from both the Australian NHMRC and ARC funding bodies and has over 90 publications. <br />
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In addition to his scientific research, Professor Phil is heavily involved in science education research. He is an Associate Fellow of the Australian Learning and Teaching Council and Research Professorial<br />
Fellow at the Centre for Educational Innovation and Technology at the University of Queensland, directed by P1. His key focus in teaching are areas around creativity and innovation and embedding transferable skills to science graduates - opportunities provided by activities such as iGEM.<br />
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Prof Phil has many recognitions of his outstanding achievements including: <br />
<br>- A UQ Foundation Research Excellence Award (2004). <br />
<br>- “In Focus” review editor for the International Journal of Biochemistry and Cell Biology, <br />
<br>- Editorial Board for the American Journal of Physiology Renal Reviews.<br />
<br>- National Secretary of the Australian and New Zealand Association for the Advancement of Science<br />
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*'''Dr Leonard Pattenden ''':<br />
[[Image:Len Wiki.jpg|right|frame]]<br />
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Dr Len Pattenden is a Senior Postdoctoral Fellow in the Poronnik group of the Health Innovations Research Institute, RMIT University. Len is the RMIT University iGEM team coordinator. Len undertakes research into albumin handling by the kidneys and the relationship of albumin structure to changes in the way it is processed by the kidneys. This research is relevant to diabetes where albumin is modified by sugars and for ~16,000 Australians progress to the complication of albuminuria, which in turn progresses to renal failure and cardiovascular disease. Len has research interests in areas of Synchrotron science, membrane biology, large-scale protein expression, protein engineering, biomolecular engineering, molecular physiology, structural biology, drug and vaccine design, and of course, synthetic biology.<br />
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'''<div style="width: 100%; height: 100%; color: #80f069; font-size: 20px;">UNDERGRADS:</div>'''<br />
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*'''Candice Raeburn''': <br />
[[Image:Halloween.jpg|right|frame]]<br />
Candi is a 2nd year biotechnology student who enjoys making surprised facial expressions at beer in conical flasks (see picture right) and genetics. When she grows up she hopes to be taller and intends to save the world (to fulfil the young optimistic scientist stereotype). Candi enjoys: bioacoustics, behavioural genetics, entomology, biological chemistry, virology, biomimicry, nanotechnology and a nice pair of slacks. She has studied in Australia and the USA, and hopes to complete her degree in the UK next year.<br />
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*'''Muriel Aldunate''':<br />
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Muriel is in her 2nd year of studying biotechnology at RMIT University and has dedicated her life to science since she was five; when her parents bought her a microscope and she went around collecting helpless insects to inspect. In her spare time she aims to inspire future generations by assisting in teaching year 7 science & being a rolemodel that can turn "geek into sheek". She plans never to stop travelling the world, marvelling at it wonders and trying to understand everything in it.<br />
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*'''Lyna Thach''':<br />
[[Image:lynathach.jpg|right|frame]]<br />
Lyna is currently studying an undergraduate degree at RMIT University; Bachelor of Applied Science (Pharmaceutical Sciences). She enjoys undertaking hands on work that involve chemicals, drugs and skilled work activities. By all means they’ve already got my best interest at heart. She decided to be part of the iGEM competition to broaden her knowledge in the laboratory field, and to have a feel of what it would be like to undertake some of the responsibilities of an Honours student. <br />
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*'''Jasmine Panthaki''':<br />
[[Image:Jazz.jpg|right|frame]]<br />
Jasmine is currently studying her first year of her undergraduate degree at RMIT University; Bachelor of Applied Science (Pharmaceutical Sciences). Her love of science developed in primary school, learning simple things about the body and space. She is determined to gain as much knowledge as she can about the mechanism of drugs and the immune system and after completing her degree she plans to travel throughout Africa.<br />
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*'''Danielle Kamato''':<br />
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Danielle Kamato is 19 years old and currently studying second year Pharmaceutical Science at RMIT university. Dannii decided to join iGEM so she would have an opportunity to research and develop a project of personal interest, as well as experience lab work first hand.<br />
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*'''Flora Yuen''':<br />
[[Image:Science.jpg|right|frame]]<br />
Flora is currently studying her first year of her undergraduate degree at RMIT University; Bachelor of Applied Science (Pharmaceutical Sciences). She has a huge passion in the field of science, although she can complain about the workload and information overload alot, deep down she loves it. She hopes that in the future that science will take her around the world. <br />
iGEM was brought to her attention since it would be a great opportunity to explore an aspect of science in great detail, while having fun.<br />
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*'''Sebastian Ramov''':<br />
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I am in my second year of Bachelor Applied science (Pharmaceutical science). I will be 21 soon and can get drunk in America lol. I like lab work because thats when everything is tied together.<br />
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*'''Breegini Culas''':<br />
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Breegini is a 1st year student studying an undergraduate degree in Applied Science (Pharmaceutical Science) at RMIT. Science has always been an interest in her life, she loves doing hands on lab work more than anything. iGEM sparked her interest because she wanted a challenge and this gave rise to a great opportunity to travel as well. <br />
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*'''Michelle Chayeb''':<br />
[[Image:michellechayeb3.jpg|right|frame]]<br />
Michelle Chayeb is a 20 year old pharmaceutical sciences student and is currently in her second year. Her primary passion lies in drug development and she's a wizz in the lab.</div>Muriel aldunate