http://2012.igem.org/wiki/index.php?title=Special:Contributions/Cpaduano&feed=atom&limit=50&target=Cpaduano&year=&month=2012.igem.org - User contributions [en]2024-03-29T05:12:04ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Cornell/project/drylab/functional_requirementsTeam:Cornell/project/drylab/functional requirements2012-10-27T02:55:06Z<p>Cpaduano: </p>
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<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Functional Requirements</h2><br />
</div><br />
</div><br />
<div><br />
<b>Scroll over each icon to find out more about the functional requirements for our device!</b><br />
</div><br />
<div class="row last-ele overflow"><br />
<div class="eight columns centered overflow" style="position:relative;"><br />
<img class="inline" src="https://static.igem.org/mediawiki/2012/6/67/Charmander_hexagon.png"><br />
<div class="func-rec-position" style="position:absolute;right:90px;top:50px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/0/09/Charmander_continuous.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Continuous</h5><br />
Pollution events can be hard to spot, making discrete testing inadequate as well as expensive. Monitoring water quality is a 24 hour job.<br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;right:-33px;top:272px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/2/2a/Charmander_field-deployable.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Field Deployable</h5><br />
Industrial water monitoring is needed in remote and rugged terrain. In order to be applicable in these terrains, our device should be durable, water-proof. Being out in the field also means all food for bacteria and power for electronics must be provided by the device itself. <br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;right:90px;bottom:25px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/e/ea/Charmander_remote.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Remote</h5><br />
To collect data in isolated locations, wireless communication is essential. This requires digital conversion of signals from our bacteria, so that it can be transmitted to the user and accessed online. <br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:90px;bottom:25px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/1/14/Charmander_electrical-output.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Electrical Output</h5><br />
Our device needs to output signal that is practical and intuitive. Electrical signal is easy to sense, process, and convert to digital signal. </div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:-35px;top:272px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/b/bb/Charmander_long-term.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Long Term</h5><br />
To monitor water quality in a truly continuous fashion, our device must be able to sustain itself without maintenance for long periods of time. Frequent maintenance is impractical.<br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:90px;top:50px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/f/f0/Charmander_safe.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Safe</h5><br />
Releasing modified organisms into the environment is a threat to both human health and biodiversity. To be field-deployable, our device must integrate multiple safeguards against escape and gene transfer.<br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
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</html></div>Cpaduanohttp://2012.igem.org/Team:Cornell/project/drylab/functional_requirementsTeam:Cornell/project/drylab/functional requirements2012-10-27T02:48:03Z<p>Cpaduano: </p>
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<h6>Dry Lab</h6><br />
</li><br />
<li class="divider"></li><br />
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<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab">How It Works</a><br />
</li><br />
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Modeling<br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/modeling/deployment">Deployment</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/modeling/time_response">Time Response</a><br />
</li><br />
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<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/status">Device Status</a><br />
</li><br />
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<a href="https://2012.igem.org/Team:Cornell/project/drylab/3dmodel">3D Model</a><br />
</li><br />
</ul><br />
</div><br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Functional Requirements</h2><br />
</div><br />
</div><br />
<div><br />
<b>Scroll over each icon to find out more about the functional requirements for our device!</b><br />
</div><br />
<div class="row last-ele overflow"><br />
<div class="eight columns centered overflow" style="position:relative;"><br />
<img class="inline" src="https://static.igem.org/mediawiki/2012/6/67/Charmander_hexagon.png"><br />
<div class="func-rec-position" style="position:absolute;right:90px;top:50px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/0/09/Charmander_continuous.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Continuous</h5><br />
Pollution events can be hard to spot, making spot testing inadequate as well as expensive. Monitoring water quality is a 24 hour job.<br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;right:-33px;top:272px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/2/2a/Charmander_field-deployable.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Field Deployable</h5><br />
Industrial water monitoring is needed in remote and rugged terrain. In order to be applicable in these terrains, our device should be durable, water-proof. Being out in the field also means all food for bacteria and power for electronics must be provided by the device itself. <br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;right:90px;bottom:25px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/e/ea/Charmander_remote.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Remote</h5><br />
To collect data in isolated locations, wireless communication is essential. This requires digital conversion of signals from our bacteria, so that it can be transmitted to the user and accessed online. <br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:90px;bottom:25px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/1/14/Charmander_electrical-output.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Electrical Output</h5><br />
Our device needs to output signal that is practical and intuitive. Electrical signal is easy to sense, process, and convert to digital signal. </div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:-35px;top:272px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/b/bb/Charmander_long-term.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Long Term</h5><br />
To monitor water quality in a truly continuous fashion, our device must be able to sustain itself without maintenance for long periods of time. Frequent maintenance is impractical.<br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:90px;top:50px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/f/f0/Charmander_safe.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Safe</h5><br />
Releasing modified organisms into the environment is a threat to both human health and biodiversity. To be field-deployable, our device must integrate multiple safeguards against escape and gene transfer.<br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<script src="https://2012.igem.org/Team:Cornell/javascripts/foundation.min?action=raw&amp;ctype=text/javascript"></script><br />
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</html></div>Cpaduanohttp://2012.igem.org/Team:Cornell/project/drylab/functional_requirementsTeam:Cornell/project/drylab/functional requirements2012-10-27T02:46:11Z<p>Cpaduano: </p>
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<li><br />
<h6>Dry Lab</h6><br />
</li><br />
<li class="divider"></li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab">How It Works</a><br />
</li><br />
<li class ="active"><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/functional_requirements">Functional Requirements</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/components">Components</a><br />
</li><br />
<li><br />
Modeling<br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/modeling/deployment">Deployment</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/modeling/time_response">Time Response</a><br />
</li><br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/status">Device Status</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/3dmodel">3D Model</a><br />
</li><br />
</ul><br />
</div><br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Functional Requirements</h2><br />
</div><br />
</div><br />
<div><br />
<b>Scroll over each icon to find out more about the functional requirements for our device!</b><br />
</div><br />
<div class="row last-ele overflow"><br />
<div class="eight columns centered overflow" style="position:relative;"><br />
<img class="inline" src="https://static.igem.org/mediawiki/2012/6/67/Charmander_hexagon.png"><br />
<div class="func-rec-position" style="position:absolute;right:90px;top:50px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/0/09/Charmander_continuous.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Continuous</h5><br />
Pollution events can be hard to spot, making spot testing inadequate as well as expensive. Monitoring water quality is a 24 hour job.<br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;right:-33px;top:272px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/2/2a/Charmander_field-deployable.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Field Deployable</h5><br />
Industrial water monitoring is needed in remote and rugged terrain. In order to be applicable in these terrains, our device should be durable, water-proof. Being out in the field also means all food for bacteria and power for electronics must be provided by the device itself. <br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;right:90px;bottom:25px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/e/ea/Charmander_remote.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Remote</h5><br />
To collect data in isolated locations, wireless communication is essential. This requires digital conversion of signals from our bacteria, so that it can be transmitted to the user and accessed online. <br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:90px;bottom:25px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/1/14/Charmander_electrical-output.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Electrical Output</h5><br />
Learn how SAFE BET communicates between bacteria and machine!<br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:-35px;top:272px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/b/bb/Charmander_long-term.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Long Term</h5><br />
To monitor water quality in a truly continuous fashion, our device must be able to sustain itself without maintenance for long periods of time. Frequent maintenance is impractical.<br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:90px;top:50px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/f/f0/Charmander_safe.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Safe</h5><br />
Releasing modified organisms into the environment is a threat to both human health and biodiversity. To be field-deployable, our device must integrate multiple safeguards against escape and gene transfer.<br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<script src="https://2012.igem.org/Team:Cornell/javascripts/foundation.min?action=raw&amp;ctype=text/javascript"></script><br />
<script src="https://2012.igem.org/Team:Cornell/javascripts/app?action=raw&amp;ctype=text/javascript"></script><br />
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<script type="text/javascript"><br />
$(window).load(function() {<br />
$('li.pg-project_drylab').addClass('active');<br />
});<br />
</script><br />
</body><br />
</html></div>Cpaduanohttp://2012.igem.org/Team:Cornell/project/drylab/functional_requirementsTeam:Cornell/project/drylab/functional requirements2012-10-27T02:44:28Z<p>Cpaduano: </p>
<hr />
<div>{{:Team:Cornell/templates/header}} <!-- paulirish.com/2008/conditional-stylesheets-vs-css-hacks-answer-neither/ --><br />
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<!--[if gt IE 8]><!--><br />
<html class="no-js" lang="en"><br />
<!--<![endif]--><br />
<div class="row"><br />
<div class="two columns"><br />
<ul class="side-nav"><br />
<li><br />
<h6>Dry Lab</h6><br />
</li><br />
<li class="divider"></li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab">How It Works</a><br />
</li><br />
<li class ="active"><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/functional_requirements">Functional Requirements</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/components">Components</a><br />
</li><br />
<li><br />
Modeling<br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/modeling/deployment">Deployment</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/modeling/time_response">Time Response</a><br />
</li><br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/status">Device Status</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/3dmodel">3D Model</a><br />
</li><br />
</ul><br />
</div><br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Functional Requirements</h2><br />
</div><br />
</div><br />
<div><br />
<b>Scroll over each icon to find out more about the functional requirements of our device!</b><br />
</div><br />
<div class="row last-ele overflow"><br />
<div class="eight columns centered overflow" style="position:relative;"><br />
<img class="inline" src="https://static.igem.org/mediawiki/2012/6/67/Charmander_hexagon.png"><br />
<div class="func-rec-position" style="position:absolute;right:90px;top:50px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/0/09/Charmander_continuous.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Continuous</h5><br />
Pollution events can be hard to spot, making spot testing inadequate as well as expensive. Monitoring water quality is a 24 hour job.<br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;right:-33px;top:272px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/2/2a/Charmander_field-deployable.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Field Deployable</h5><br />
Industrial water monitoring is needed in remote and rugged terrain. In order to be applicable in these terrains, our device should be durable, water-proof. Being out in the field also means all food for bacteria and power for electronics must be provided by the device itself. <br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;right:90px;bottom:25px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/e/ea/Charmander_remote.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Remote</h5><br />
To collect data in isolated locations, wireless communication is essential. This requires digital conversion of signals from our bacteria, so that it can be transmitted to the user and accessed online. <br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:90px;bottom:25px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/1/14/Charmander_electrical-output.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Electrical Output</h5><br />
Learn how SAFE BET communicates between bacteria and machine!<br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:-35px;top:272px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/b/bb/Charmander_long-term.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Long Term</h5><br />
To monitor water quality in a truly continuous fashion, our device must be able to sustain itself without maintenance for long periods of time. Frequent maintenance is impractical.<br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:90px;top:50px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/f/f0/Charmander_safe.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Safe</h5><br />
Releasing modified organisms into the environment is a threat to both human health and biodiversity. To be field-deployable, our device must integrate multiple safeguards against escape and gene transfer.<br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
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</html></div>Cpaduanohttp://2012.igem.org/Team:Cornell/project/drylab/functional_requirementsTeam:Cornell/project/drylab/functional requirements2012-10-27T02:42:58Z<p>Cpaduano: </p>
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<div class="row"><br />
<div class="two columns"><br />
<ul class="side-nav"><br />
<li><br />
<h6>Dry Lab</h6><br />
</li><br />
<li class="divider"></li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab">How It Works</a><br />
</li><br />
<li class ="active"><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/functional_requirements">Functional Requirements</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/components">Components</a><br />
</li><br />
<li><br />
Modeling<br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/modeling/deployment">Deployment</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/modeling/time_response">Time Response</a><br />
</li><br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/status">Device Status</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/drylab/3dmodel">3D Model</a><br />
</li><br />
</ul><br />
</div><br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Functional Requirements</h2><br />
</div><br />
</div><br />
<div class="row last-ele overflow"><br />
<div class="eight columns centered overflow" style="position:relative;"><br />
<img class="inline" src="https://static.igem.org/mediawiki/2012/6/67/Charmander_hexagon.png"><br />
<div class="func-rec-position" style="position:absolute;right:90px;top:50px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/0/09/Charmander_continuous.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Continuous</h5><br />
Pollution events can be hard to spot, making spot testing inadequate as well as expensive. Monitoring water quality is a 24 hour job.<br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;right:-33px;top:272px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/2/2a/Charmander_field-deployable.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Field Deployable</h5><br />
Industrial water monitoring is needed in remote and rugged terrain. In order to be applicable in these terrains, our device should be durable, water-proof. Being out in the field also means all food for bacteria and power for electronics must be provided by the device itself. <br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;right:90px;bottom:25px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/e/ea/Charmander_remote.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Remote</h5><br />
To collect data in isolated locations, wireless communication is essential. This requires digital conversion of signals from our bacteria, so that it can be transmitted to the user and accessed online. <br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:90px;bottom:25px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/1/14/Charmander_electrical-output.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Electrical Output</h5><br />
Learn how SAFE BET communicates between bacteria and machine!<br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:-35px;top:272px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/b/bb/Charmander_long-term.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Long Term</h5><br />
To monitor water quality in a truly continuous fashion, our device must be able to sustain itself without maintenance for long periods of time. Frequent maintenance is impractical.<br />
</div><br />
</div><br />
<div class="func-rec-position" style="position:absolute;left:90px;top:50px;"><br />
<div class="func-req-hover" ><br />
<img src="https://static.igem.org/mediawiki/2012/f/f0/Charmander_safe.png"><br />
</div><br />
<div class="func-req-desc"><br />
<h5>Safe</h5><br />
Releasing modified organisms into the environment is a threat to both human health and biodiversity. To be field-deployable, our device must integrate multiple safeguards against escape and gene transfer.<br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<script src="https://2012.igem.org/Team:Cornell/javascripts/foundation.min?action=raw&amp;ctype=text/javascript"></script><br />
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</html></div>Cpaduanohttp://2012.igem.org/Team:Cornell/project/wetlab/assemblyTeam:Cornell/project/wetlab/assembly2012-10-27T02:26:13Z<p>Cpaduano: </p>
<hr />
<div>{{:Team:Cornell/templates/header}} <!-- paulirish.com/2008/conditional-stylesheets-vs-css-hacks-answer-neither/ --><br />
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<html class="no-js" lang="en"><br />
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<div class="row"><br />
<div class="two columns"><br />
<ul class="side-nav"><br />
<li><br />
<h6>Wet Lab</h6><br />
</li><br />
<li class="divider"></li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/chassis">Chassis</a><br />
</li><br />
<li class="active"><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly">DNA Assembly</a></li><li><br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/arsenic">Arsenic Reporter</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/naphthalene">Naphthalene Reporter</a><br />
</li><br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results">Testing &amp; Results</a> <br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/biobricks">BioBricks</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</a><br />
</li> <br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/nah_operon"><i>nah</i> Operon Expression</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/artificial_river_media">Artificial River Media</a><br />
</li><br />
<br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/animation">Animation</a><br />
</li><br />
</ul><br />
</div><br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">DNA Assembly</h2><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="six columns"><br />
<img class="inline" src="https://static.igem.org/mediawiki/2012/e/e5/SWITCHOFF.png" width="550" height="600"><br />
<br><b>When MtrB is not present, it is as if the switch is open, and current cannot flow.</b><br />
</div><br />
<div class="six columns"><br />
<i>Shewanella oneidensis</i> MR-1’s metal reduction pathway can be likened to a simple switch analogy. Without MtrB, no extracellular transfer of electrons is possible, and therefore no current is produced. However, when reintroduced, MtrB closes the switch therefore allowing extracellular reduction and current generation.<br />
<br><br><br />
<br />
<br />
To sensitize <i>Shewanella’s</i> metal reduction pathway to our analytes, we decided to use a complementation strategy. By using the <i>Shewanella</i> MtrB knockout strain, JG 700, which was graciously provided by Professor Jeffery Gralnick from the University of Minnesota, we are able to reintroduce MtrB on a plasmid under the control of inducible promoters sensitive to the analytes we want to detect. Thus, MtrB&#8212;and therefore current&#8212;should only be produced in the presence of analyte.<br><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="six columns"><br />
<img class="inline" src="https://static.igem.org/mediawiki/2012/a/a5/SWITCHON.png" width="550" height="600"><br />
<br><b>When MtrB is reintroduced into the system, it is as if the switch is closed, allowing current to flow.</b><br />
</div><br />
<div class="six columns"><br />
<br>One of the greatest strengths of this approach is its modularity; by simply switching out the sensing region on the plasmid, we can sensitize MtrB production to any analyte for which genetic parts exist. <br><br><br />
<br />
Because of its essential role in extracellular electron transfer, MtrB can be thought of as an electric switch: When absent, the switch is open, disallowing current production in microbial electrochemical systems; when present, the switch is closed and current may be produced. We took advantage of this in the development of our biosensing strains: Using a &Delta;mtrB strain (JG700) as a host, we adopted a complementation strategy wherein the capability for extracellular electron shuttling was reintroduced via the expression of MtrB from a plasmid. Because we designed our engineered plasmids so that mtrB transcription could be induced in response to our analyte of interest, we were able to construct strains that produce more current in response to analyte—as the MtrB ‘switches’ close. <br />
</div><br />
<br />
</div><br />
</div><br />
</div><br />
<script src="https://2012.igem.org/Team:Cornell/javascripts/foundation.min?action=raw&amp;ctype=text/javascript"></script><br />
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</html></div>Cpaduanohttp://2012.igem.org/Team:Cornell/project/wetlab/assemblyTeam:Cornell/project/wetlab/assembly2012-10-27T02:15:01Z<p>Cpaduano: </p>
<hr />
<div>{{:Team:Cornell/templates/header}} <!-- paulirish.com/2008/conditional-stylesheets-vs-css-hacks-answer-neither/ --><br />
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<html class="no-js" lang="en"><br />
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<div class="row"><br />
<div class="two columns"><br />
<ul class="side-nav"><br />
<li><br />
<h6>Wet Lab</h6><br />
</li><br />
<li class="divider"></li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/chassis">Chassis</a><br />
</li><br />
<li class="active"><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly">DNA Assembly</a></li><li><br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/arsenic">Arsenic Reporter</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/naphthalene">Naphthalene Reporter</a><br />
</li><br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results">Testing &amp; Results</a> <br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/biobricks">BioBricks</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</a><br />
</li> <br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/nah_operon"><i>nah</i> Operon Expression</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/artificial_river_media">Artificial River Media</a><br />
</li><br />
<br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/animation">Animation</a><br />
</li><br />
</ul><br />
</div><br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">DNA Assembly</h2><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="six columns"><br />
<img class="inline" src="https://static.igem.org/mediawiki/2012/e/e5/SWITCHOFF.png" width="550" height="600"><br />
<br><b>When MtrB is not present, it is as if the switch is open, and current cannot flow.</b><br />
</div><br />
<div class="six columns"><br />
<i>Shewanella oneidensis</i> MR-1’s metal reduction pathway can be likened to a simple switch analogy. Without MtrB, no extracellular transfer of electrons is possible, and therefore no current is produced. However, when reintroduced, MtrB closes the switch therefore allowing extracellular reduction and current generation.<br />
<br><br><br />
<br />
<br />
To sensitize <i>Shewanella’s</i> metal reduction pathway to our analytes, we decided to use a complementation strategy. By using the <i>Shewanella</i> MtrB knockout strain, JG 700, which was graciously provided by Professor Jeffery Gralnick from the University of Minnesota, we are able to reintroduce MtrB on a plasmid under the control of inducible promoters sensitive to the analytes we want to detect. Thus, MtrB&#8212;and therefore current&#8212;should only be produced in the presence of analyte.<br><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="six columns"><br />
<img class="inline" src="https://static.igem.org/mediawiki/2012/a/a5/SWITCHON.png" width="550" height="600"><br />
<br><b>When MtrB is reintroduced into the system, it is as if the switch is closed, allowing current to flow.</b><br />
</div><br />
<div class="six columns"><br />
<br>One of the greatest strengths of this approach is its modularity; by simply switching out the sensing region on the plasmid, we can sensitize MtrB production to any analyte for which genetic parts exist. <br><br><br />
<br />
Because of its essential role in extracellular electron transfer, MtrB can be thought of as an electric switch: When absent, the switch is open, disallowing current production in microbial electrochemical systems; when present, the switch is closed and current may be produced. We took advantage of this in the development of our biosensing strains: Using a dmtrB strain (JG700) as a host, we adopted a complementation strategy wherein the capability for extracellular electron shuttling was reintroduced via the expression of MtrB from a plasmid. Because we designed our engineered plasmids so that mtrB transcription could be induced in response to our analyte of interest, we were able to construct strains that produce more current in response to analyte—as the MtrB ‘switches’ close. <br />
</div><br />
<br />
</div><br />
</div><br />
</div><br />
<script src="https://2012.igem.org/Team:Cornell/javascripts/foundation.min?action=raw&amp;ctype=text/javascript"></script><br />
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</html></div>Cpaduanohttp://2012.igem.org/Team:Cornell/project/wetlab/assemblyTeam:Cornell/project/wetlab/assembly2012-10-27T02:13:50Z<p>Cpaduano: </p>
<hr />
<div>{{:Team:Cornell/templates/header}} <!-- paulirish.com/2008/conditional-stylesheets-vs-css-hacks-answer-neither/ --><br />
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<html class="no-js" lang="en"><br />
<!--<![endif]--><br />
<div class="row"><br />
<div class="two columns"><br />
<ul class="side-nav"><br />
<li><br />
<h6>Wet Lab</h6><br />
</li><br />
<li class="divider"></li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/chassis">Chassis</a><br />
</li><br />
<li class="active"><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly">DNA Assembly</a></li><li><br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/arsenic">Arsenic Reporter</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/naphthalene">Naphthalene Reporter</a><br />
</li><br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results">Testing &amp; Results</a> <br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/biobricks">BioBricks</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</a><br />
</li> <br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/nah_operon"><i>nah</i> Operon Expression</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/artificial_river_media">Artificial River Media</a><br />
</li><br />
<br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/animation">Animation</a><br />
</li><br />
</ul><br />
</div><br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">DNA Assembly</h2><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="six columns"><br />
<img class="inline" src="https://static.igem.org/mediawiki/2012/e/e5/SWITCHOFF.png" width="550" height="600"><br />
</div><br />
<div class="six columns"><br />
<i>Shewanella oneidensis</i> MR-1’s metal reduction pathway can be likened to a simple switch analogy. Without MtrB, no extracellular transfer of electrons is possible, and therefore no current is produced. However, when reintroduced, MtrB closes the switch therefore allowing extracellular reduction and current generation.<br />
<br><br><br />
<br />
<br />
To sensitize <i>Shewanella’s</i> metal reduction pathway to our analytes, we decided to use a complementation strategy. By using the <i>Shewanella</i> MtrB knockout strain, JG 700, which was graciously provided by Professor Jeffery Gralnick from the University of Minnesota, we are able to reintroduce MtrB on a plasmid under the control of inducible promoters sensitive to the analytes we want to detect. Thus, MtrB&#8212;and therefore current&#8212;should only be produced in the presence of analyte.<br><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="six columns"><br />
<img class="inline" src="https://static.igem.org/mediawiki/2012/a/a5/SWITCHON.png" width="550" height="600"><br />
</div><br />
<div class="six columns"><br />
<br>One of the greatest strengths of this approach is its modularity; by simply switching out the sensing region on the plasmid, we can sensitize MtrB production to any analyte for which genetic parts exist. <br><br><br />
<br />
Because of its essential role in extracellular electron transfer, MtrB can be thought of as an electric switch: When absent, the switch is open, disallowing current production in microbial electrochemical systems; when present, the switch is closed and current may be produced. We took advantage of this in the development of our biosensing strains: Using a dmtrB strain (JG700) as a host, we adopted a complementation strategy wherein the capability for extracellular electron shuttling was reintroduced via the expression of MtrB from a plasmid. Because we designed our engineered plasmids so that mtrB transcription could be induced in response to our analyte of interest, we were able to construct strains that produce more current in response to analyte—as the MtrB ‘switches’ close. <br />
</div><br />
<br />
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<div></div>Cpaduanohttp://2012.igem.org/Team:Cornell/project/wetlab/resultsTeam:Cornell/project/wetlab/results2012-10-27T01:21:29Z<p>Cpaduano: </p>
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly">DNA Assembly</a><br />
<ul><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/arsenic">Arsenic Reporter</a><br />
</li><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/naphthalene">Naphthalene Reporter</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a><br />
</li><br />
<br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/nah_operon"><i>nah</i> Operon Expression</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/artificial_river_media">Artificial River Media</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
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<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Results</h2><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/a/a7/FluorescenceArsenite1001.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/2/2b/Arsenite_300.png"></a><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/5/56/FluorescenceSalicylate930.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/e/e2/Salycilate_300.png"></a><br />
</div><br />
<div class="nine columns"><br />
<h3>Transcriptional Characterization</h3><br />
After constructing our reporters, we first wanted to verify that they operated on a transcriptional level in <i>S. oneidensis</i>. Our transcriptional characterization consisted of fluorescence testing and RT-qPCR. <br />
<br><br><br />
In order to conduct fluorescence characterization, we appended mRFP downstream of mtrB in our arsenic and salicylate reporters. We then measured the relative fluorescence of the reporters when induced with different concentrations of arsenic and salicylate, respectively. Both showed increased fluorescence in response to increased analyte, strongly suggesting transcriptional upregulation of mRFP and therefore of upstream mtrB. <br />
<br><br><br />
We are also in the process of conducting real-time quantitative PCR, as a more direct method of demonstrating transcriptional upregulation of mtrB in response to analyte.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3>Current Response</h3><br />
Encouraged by our positive results from fluorescence characterization, we moved forward with testing our reporter strains in bioreactors. We demonstrated that our arsenic reporter strain functions at the system level: current is upregulated in response to increasing arsenite concentration. However, leaky expression in our salicylate reporter strain saturated current response at basal levels, an issue we plan to address in the future.<br />
<br><br><br />
As a proxy to current production, we are also conducting ferrozine assays, which measure how much iron (III) is reduced to iron (II) by extracellular electron shuttling. This is a high-throughput method of assessing the relative functionality of our reporter strains at a system level.<br />
</div><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/b/bf/Currentresponse_600.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/2/2e/Currentresponse_300.png"></a><br />
</div><br />
</div> <br />
<div class="row"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/d/dd/Smallworker.png"><br />
</div><br />
<div class="nine columns"><br />
<h3>MtrB Protein Expression</h3><br />
In addition to characterization at the transcriptional and whole-system levels, we also want to verify that MtrB, our protein of interest, is being expressed at higher levels when the reporters are induced with analyte. To this end, we are in the process of conducting Western blots. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3><i>nah</i> Operon Expression</h3><br />
To test the functionality of our third major construct, the <i>nah</i> operon, we are currently conducting tests on the biosynthesis of indigo by naphthalene dioxygenase, an enzyme encoded in the <i>nah</i> operon. <br />
</div><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/a/a1/IndigoReaction.jpg" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/4/48/Indigo.png"></a><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/a/ac/Sal1GrowthCurve.jpg" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/0/01/Growth_300.png"></a><br />
</div><br />
<div class="nine columns"><br />
<h3>Artificial River Media</h3><br />
As a practical consideration for the deployment of our device, we attempted to develop a minimal growth medium that could be highly concentrated in the food tanks of our final device, and fed to the cells at a low flow rate. Our results suggest that our strains could be sustained on a steady flow of 2% (w/v) sodium lactate solution. <br />
</div><br />
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</html></div>Cpaduanohttp://2012.igem.org/File:Smallworker.pngFile:Smallworker.png2012-10-27T01:21:12Z<p>Cpaduano: </p>
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<div></div>Cpaduanohttp://2012.igem.org/Team:Cornell/project/wetlab/resultsTeam:Cornell/project/wetlab/results2012-10-27T01:20:10Z<p>Cpaduano: </p>
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<h6>Wet Lab</h6><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/chassis">Chassis</a><br />
</li><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly">DNA Assembly</a><br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/arsenic">Arsenic Reporter</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/naphthalene">Naphthalene Reporter</a><br />
</li><br />
</ul><br />
</li><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results">Testing &amp; Results</a></li><li> <br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/biobricks">BioBricks</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/nah_operon"><i>nah</i> Operon Expression</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/artificial_river_media">Artificial River Media</a><br />
</li><br />
<br />
</ul><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
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</li><br />
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<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Results</h2><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/a/a7/FluorescenceArsenite1001.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/2/2b/Arsenite_300.png"></a><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/5/56/FluorescenceSalicylate930.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/e/e2/Salycilate_300.png"></a><br />
</div><br />
<div class="nine columns"><br />
<h3>Transcriptional Characterization</h3><br />
After constructing our reporters, we first wanted to verify that they operated on a transcriptional level in <i>S. oneidensis</i>. Our transcriptional characterization consisted of fluorescence testing and RT-qPCR. <br />
<br><br><br />
In order to conduct fluorescence characterization, we appended mRFP downstream of mtrB in our arsenic and salicylate reporters. We then measured the relative fluorescence of the reporters when induced with different concentrations of arsenic and salicylate, respectively. Both showed increased fluorescence in response to increased analyte, strongly suggesting transcriptional upregulation of mRFP and therefore of upstream mtrB. <br />
<br><br><br />
We are also in the process of conducting real-time quantitative PCR, as a more direct method of demonstrating transcriptional upregulation of mtrB in response to analyte.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3>Current Response</h3><br />
Encouraged by our positive results from fluorescence characterization, we moved forward with testing our reporter strains in bioreactors. We demonstrated that our arsenic reporter strain functions at the system level: current is upregulated in response to increasing arsenite concentration. However, leaky expression in our salicylate reporter strain saturated current response at basal levels, an issue we plan to address in the future.<br />
<br><br><br />
As a proxy to current production, we are also conducting ferrozine assays, which measure how much iron (III) is reduced to iron (II) by extracellular electron shuttling. This is a high-throughput method of assessing the relative functionality of our reporter strains at a system level.<br />
</div><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/b/bf/Currentresponse_600.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/2/2e/Currentresponse_300.png"></a><br />
</div><br />
</div> <br />
<div class="row"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/2/26/Cornell12_Stock_10.jpg"><br />
</div><br />
<div class="nine columns"><br />
<h3>MtrB Protein Expression</h3><br />
In addition to characterization at the transcriptional and whole-system levels, we also want to verify that MtrB, our protein of interest, is being expressed at higher levels when the reporters are induced with analyte. To this end, we are in the process of conducting Western blots. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3><i>nah</i> Operon Expression</h3><br />
To test the functionality of our third major construct, the <i>nah</i> operon, we are currently conducting tests on the biosynthesis of indigo by naphthalene dioxygenase, an enzyme encoded in the <i>nah</i> operon. <br />
</div><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/a/a1/IndigoReaction.jpg" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/4/48/Indigo.png"></a><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/a/ac/Sal1GrowthCurve.jpg" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/0/01/Growth_300.png"></a><br />
</div><br />
<div class="nine columns"><br />
<h3>Artificial River Media</h3><br />
As a practical consideration for the deployment of our device, we attempted to develop a minimal growth medium that could be highly concentrated in the food tanks of our final device, and fed to the cells at a low flow rate. Our results suggest that our strains could be sustained on a steady flow of 2% (w/v) sodium lactate solution. <br />
</div><br />
</div><br />
</div><br />
</div><br />
<script src="https://2012.igem.org/Team:Cornell/javascripts/foundation.min?action=raw&amp;ctype=text/javascript"></script><br />
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</html></div>Cpaduanohttp://2012.igem.org/Team:Cornell/project/wetlab/resultsTeam:Cornell/project/wetlab/results2012-10-27T01:19:06Z<p>Cpaduano: </p>
<hr />
<div>{{:Team:Cornell/templates/header}} <!-- paulirish.com/2008/conditional-stylesheets-vs-css-hacks-answer-neither/ --><br />
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<li><br />
<h6>Wet Lab</h6><br />
</li><br />
<li class="divider"></li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/chassis">Chassis</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly">DNA Assembly</a><br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/arsenic">Arsenic Reporter</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/naphthalene">Naphthalene Reporter</a><br />
</li><br />
</ul><br />
</li><br />
<li class="active"><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results">Testing &amp; Results</a></li><li> <br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/biobricks">BioBricks</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/nah_operon"><i>nah</i> Operon Expression</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/artificial_river_media">Artificial River Media</a><br />
</li><br />
<br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/animation">Animation</a><br />
</li><br />
</ul><br />
</div><br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Results</h2><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/a/a7/FluorescenceArsenite1001.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/2/2b/Arsenite_300.png"></a><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/5/56/FluorescenceSalicylate930.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/e/e2/Salycilate_300.png"></a><br />
</div><br />
<div class="nine columns"><br />
<h3>Transcriptional Characterization</h3><br />
After constructing our reporters, we first wanted to verify that they operated on a transcriptional level in <i>S. oneidensis</i>. Our transcriptional characterization consisted of fluorescence testing and RT-qPCR. <br />
<br><br><br />
In order to conduct fluorescence characterization, we appended mRFP downstream of mtrB in our arsenic and salicylate reporters. We then measured the relative fluorescence of the reporters when induced with different concentrations of arsenic and salicylate, respectively. Both showed increased fluorescence in response to increased analyte, strongly suggesting transcriptional upregulation of mRFP and therefore of upstream mtrB. <br />
<br><br><br />
We are also in the process of conducting real-time quantitative PCR, as a more direct method of demonstrating transcriptional upregulation of mtrB in response to analyte.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3>Current Response</h3><br />
Encouraged by our positive results from fluorescence characterization, we moved forward with testing our reporter strains in bioreactors. We demonstrated that our arsenic reporter strain functions at the system level: current is upregulated in response to increasing arsenite concentration. However, leaky expression in our salicylate reporter strain saturated current response at basal levels, an issue we plan to address in the future.<br />
<br><br><br />
As a proxy to current production, we are also conducting ferrozine assays, which measure how much iron (III) is reduced to iron (II) by extracellular electron shuttling. This is a high-throughput method of assessing the relative functionality of our reporter strains at a system level.<br />
</div><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/b/bf/Currentresponse_600.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/2/2e/Currentresponse_300.png"></a><br />
</div><br />
</div> <br />
<div class="row"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/4/43/Syntheticriver.jpg"><br />
</div><br />
<div class="nine columns"><br />
<h3>MtrB Protein Expression</h3><br />
In addition to characterization at the transcriptional and whole-system levels, we also want to verify that MtrB, our protein of interest, is being expressed at higher levels when the reporters are induced with analyte. To this end, we are in the process of conducting Western blots. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3><i>nah</i> Operon Expression</h3><br />
To test the functionality of our third major construct, the <i>nah</i> operon, we are currently conducting tests on the biosynthesis of indigo by naphthalene dioxygenase, an enzyme encoded in the <i>nah</i> operon. <br />
</div><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/a/a1/IndigoReaction.jpg" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/4/48/Indigo.png"></a><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/a/ac/Sal1GrowthCurve.jpg" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/0/01/Growth_300.png"></a><br />
</div><br />
<div class="nine columns"><br />
<h3>Artificial River Media</h3><br />
As a practical consideration for the deployment of our device, we attempted to develop a minimal growth medium that could be highly concentrated in the food tanks of our final device, and fed to the cells at a low flow rate. Our results suggest that our strains could be sustained on a steady flow of 2% (w/v) sodium lactate solution. <br />
</div><br />
</div><br />
</div><br />
</div><br />
<script src="https://2012.igem.org/Team:Cornell/javascripts/foundation.min?action=raw&amp;ctype=text/javascript"></script><br />
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</html></div>Cpaduanohttp://2012.igem.org/File:Growth_300.pngFile:Growth 300.png2012-10-27T01:18:45Z<p>Cpaduano: </p>
<hr />
<div></div>Cpaduanohttp://2012.igem.org/Team:Cornell/project/wetlab/resultsTeam:Cornell/project/wetlab/results2012-10-27T01:17:35Z<p>Cpaduano: </p>
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/chassis">Chassis</a><br />
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<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/arsenic">Arsenic Reporter</a><br />
</li><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/naphthalene">Naphthalene Reporter</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results">Testing &amp; Results</a></li><li> <br />
<ul><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/biobricks">BioBricks</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</a><br />
</li><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/nah_operon"><i>nah</i> Operon Expression</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/artificial_river_media">Artificial River Media</a><br />
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<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Results</h2><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/a/a7/FluorescenceArsenite1001.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/2/2b/Arsenite_300.png"></a><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/5/56/FluorescenceSalicylate930.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/e/e2/Salycilate_300.png"></a><br />
</div><br />
<div class="nine columns"><br />
<h3>Transcriptional Characterization</h3><br />
After constructing our reporters, we first wanted to verify that they operated on a transcriptional level in <i>S. oneidensis</i>. Our transcriptional characterization consisted of fluorescence testing and RT-qPCR. <br />
<br><br><br />
In order to conduct fluorescence characterization, we appended mRFP downstream of mtrB in our arsenic and salicylate reporters. We then measured the relative fluorescence of the reporters when induced with different concentrations of arsenic and salicylate, respectively. Both showed increased fluorescence in response to increased analyte, strongly suggesting transcriptional upregulation of mRFP and therefore of upstream mtrB. <br />
<br><br><br />
We are also in the process of conducting real-time quantitative PCR, as a more direct method of demonstrating transcriptional upregulation of mtrB in response to analyte.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3>Current Response</h3><br />
Encouraged by our positive results from fluorescence characterization, we moved forward with testing our reporter strains in bioreactors. We demonstrated that our arsenic reporter strain functions at the system level: current is upregulated in response to increasing arsenite concentration. However, leaky expression in our salicylate reporter strain saturated current response at basal levels, an issue we plan to address in the future.<br />
<br><br><br />
As a proxy to current production, we are also conducting ferrozine assays, which measure how much iron (III) is reduced to iron (II) by extracellular electron shuttling. This is a high-throughput method of assessing the relative functionality of our reporter strains at a system level.<br />
</div><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/b/bf/Currentresponse_600.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/2/2e/Currentresponse_300.png"></a><br />
</div><br />
</div> <br />
<div class="row"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/4/43/Syntheticriver.jpg"><br />
</div><br />
<div class="nine columns"><br />
<h3>MtrB Protein Expression</h3><br />
In addition to characterization at the transcriptional and whole-system levels, we also want to verify that MtrB, our protein of interest, is being expressed at higher levels when the reporters are induced with analyte. To this end, we are in the process of conducting Western blots. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3><i>nah</i> Operon Expression</h3><br />
To test the functionality of our third major construct, the <i>nah</i> operon, we are currently conducting tests on the biosynthesis of indigo by naphthalene dioxygenase, an enzyme encoded in the <i>nah</i> operon. <br />
</div><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/a/a1/IndigoReaction.jpg" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/4/48/Indigo.png"></a><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/4/43/Syntheticriver.jpg"><br />
</div><br />
<div class="nine columns"><br />
<h3>Artificial River Media</h3><br />
As a practical consideration for the deployment of our device, we attempted to develop a minimal growth medium that could be highly concentrated in the food tanks of our final device, and fed to the cells at a low flow rate. Our results suggest that our strains could be sustained on a steady flow of 2% (w/v) sodium lactate solution. <br />
</div><br />
</div><br />
</div><br />
</div><br />
<script src="https://2012.igem.org/Team:Cornell/javascripts/foundation.min?action=raw&amp;ctype=text/javascript"></script><br />
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</html></div>Cpaduanohttp://2012.igem.org/File:Indigo.pngFile:Indigo.png2012-10-27T01:17:23Z<p>Cpaduano: </p>
<hr />
<div></div>Cpaduanohttp://2012.igem.org/Team:Cornell/project/wetlab/resultsTeam:Cornell/project/wetlab/results2012-10-27T01:12:56Z<p>Cpaduano: </p>
<hr />
<div>{{:Team:Cornell/templates/header}} <!-- paulirish.com/2008/conditional-stylesheets-vs-css-hacks-answer-neither/ --><br />
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<div class="row"><br />
<div class="two columns"><br />
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<li><br />
<h6>Wet Lab</h6><br />
</li><br />
<li class="divider"></li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/chassis">Chassis</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly">DNA Assembly</a><br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/arsenic">Arsenic Reporter</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/naphthalene">Naphthalene Reporter</a><br />
</li><br />
</ul><br />
</li><br />
<li class="active"><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results">Testing &amp; Results</a></li><li> <br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/biobricks">BioBricks</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/nah_operon"><i>nah</i> Operon Expression</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/artificial_river_media">Artificial River Media</a><br />
</li><br />
<br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
</li><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/animation">Animation</a><br />
</li><br />
</ul><br />
</div><br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Results</h2><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/a/a7/FluorescenceArsenite1001.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/2/2b/Arsenite_300.png"></a><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/5/56/FluorescenceSalicylate930.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/e/e2/Salycilate_300.png"></a><br />
</div><br />
<div class="nine columns"><br />
<h3>Transcriptional Characterization</h3><br />
After constructing our reporters, we first wanted to verify that they operated on a transcriptional level in <i>S. oneidensis</i>. Our transcriptional characterization consisted of fluorescence testing and RT-qPCR. <br />
<br><br><br />
In order to conduct fluorescence characterization, we appended mRFP downstream of mtrB in our arsenic and salicylate reporters. We then measured the relative fluorescence of the reporters when induced with different concentrations of arsenic and salicylate, respectively. Both showed increased fluorescence in response to increased analyte, strongly suggesting transcriptional upregulation of mRFP and therefore of upstream mtrB. <br />
<br><br><br />
We are also in the process of conducting real-time quantitative PCR, as a more direct method of demonstrating transcriptional upregulation of mtrB in response to analyte.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3>Current Response</h3><br />
Encouraged by our positive results from fluorescence characterization, we moved forward with testing our reporter strains in bioreactors. We demonstrated that our arsenic reporter strain functions at the system level: current is upregulated in response to increasing arsenite concentration. However, leaky expression in our salicylate reporter strain saturated current response at basal levels, an issue we plan to address in the future.<br />
<br><br><br />
As a proxy to current production, we are also conducting ferrozine assays, which measure how much iron (III) is reduced to iron (II) by extracellular electron shuttling. This is a high-throughput method of assessing the relative functionality of our reporter strains at a system level.<br />
</div><br />
<div class="three columns"><br />
<a href="https://static.igem.org/mediawiki/2012/b/bf/Currentresponse_600.png" rel="lightbox[project-page]"><img src="https://static.igem.org/mediawiki/2012/2/2e/Currentresponse_300.png"></a><br />
</div><br />
</div> <br />
<div class="row"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/4/43/Syntheticriver.jpg"><br />
</div><br />
<div class="nine columns"><br />
<h3>MtrB Protein Expression</h3><br />
In addition to characterization at the transcriptional and whole-system levels, we also want to verify that MtrB, our protein of interest, is being expressed at higher levels when the reporters are induced with analyte. To this end, we are in the process of conducting Western blots. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3><i>nah</i> Operon Expression</h3><br />
To test the functionality of our third major construct, the <i>nah</i> operon, we are currently conducting tests on the biosynthesis of indigo by naphthalene dioxygenase, an enzyme encoded in the <i>nah</i> operon. <br />
</div><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/4/43/Syntheticriver.jpg"><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/4/43/Syntheticriver.jpg"><br />
</div><br />
<div class="nine columns"><br />
<h3>Artificial River Media</h3><br />
As a practical consideration for the deployment of our device, we attempted to develop a minimal growth medium that could be highly concentrated in the food tanks of our final device, and fed to the cells at a low flow rate. Our results suggest that our strains could be sustained on a steady flow of 2% (w/v) sodium lactate solution. <br />
</div><br />
</div><br />
</div><br />
</div><br />
<script src="https://2012.igem.org/Team:Cornell/javascripts/foundation.min?action=raw&amp;ctype=text/javascript"></script><br />
<script src="https://2012.igem.org/Team:Cornell/javascripts/app?action=raw&amp;ctype=text/javascript"></script><br />
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</html></div>Cpaduanohttp://2012.igem.org/File:Salycilate_300.pngFile:Salycilate 300.png2012-10-27T01:12:46Z<p>Cpaduano: </p>
<hr />
<div></div>Cpaduanohttp://2012.igem.org/File:Arsenite_300.pngFile:Arsenite 300.png2012-10-27T01:11:58Z<p>Cpaduano: </p>
<hr />
<div></div>Cpaduanohttp://2012.igem.org/Team:Cornell/project/wetlab/resultsTeam:Cornell/project/wetlab/results2012-10-27T01:08:39Z<p>Cpaduano: </p>
<hr />
<div>{{:Team:Cornell/templates/header}} <!-- paulirish.com/2008/conditional-stylesheets-vs-css-hacks-answer-neither/ --><br />
<!--[if lt IE 7]> <html class="no-js lt-ie9 lt-ie8 lt-ie7" lang="en"> <![endif]--><br />
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<!--<![endif]--><br />
<div class="row"><br />
<div class="two columns"><br />
<ul class="side-nav"><br />
<li><br />
<h6>Wet Lab</h6><br />
</li><br />
<li class="divider"></li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/chassis">Chassis</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly">DNA Assembly</a><br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/arsenic">Arsenic Reporter</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/naphthalene">Naphthalene Reporter</a><br />
</li><br />
</ul><br />
</li><br />
<li class="active"><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results">Testing &amp; Results</a></li><li> <br />
<ul><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/biobricks">BioBricks</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a><br />
</li><br />
<br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/nah_operon"><i>nah</i> Operon Expression</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/artificial_river_media">Artificial River Media</a><br />
</li><br />
<br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Cornell/project/wetlab/animation">Animation</a><br />
</li><br />
</ul><br />
</div><br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Results</h2><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Foodtank.jpg"><br />
</div><br />
<div class="nine columns"><br />
<h3>Transcriptional Characterization</h3><br />
After constructing our reporters, we first wanted to verify that they operated on a transcriptional level in <i>S. oneidensis</i>. Our transcriptional characterization consisted of fluorescence testing and RT-qPCR. <br />
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In order to conduct fluorescence characterization, we appended mRFP downstream of mtrB in our arsenic and salicylate reporters. We then measured the relative fluorescence of the reporters when induced with different concentrations of arsenic and salicylate, respectively. Both showed increased fluorescence in response to increased analyte, strongly suggesting transcriptional upregulation of mRFP and therefore of upstream mtrB. <br />
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We are also in the process of conducting real-time quantitative PCR, as a more direct method of demonstrating transcriptional upregulation of mtrB in response to analyte.<br />
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<h3>Current Response</h3><br />
Encouraged by our positive results from fluorescence characterization, we moved forward with testing our reporter strains in bioreactors. We demonstrated that our arsenic reporter strain functions at the system level: current is upregulated in response to increasing arsenite concentration. However, leaky expression in our salicylate reporter strain saturated current response at basal levels, an issue we plan to address in the future.<br />
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As a proxy to current production, we are also conducting ferrozine assays, which measure how much iron (III) is reduced to iron (II) by extracellular electron shuttling. This is a high-throughput method of assessing the relative functionality of our reporter strains at a system level.<br />
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<h3>MtrB Protein Expression</h3><br />
In addition to characterization at the transcriptional and whole-system levels, we also want to verify that MtrB, our protein of interest, is being expressed at higher levels when the reporters are induced with analyte. To this end, we are in the process of conducting Western blots. <br />
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<h3><i>nah</i> Operon Expression</h3><br />
To test the functionality of our third major construct, the <i>nah</i> operon, we are currently conducting tests on the biosynthesis of indigo by naphthalene dioxygenase, an enzyme encoded in the <i>nah</i> operon. <br />
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<h3>Artificial River Media</h3><br />
As a practical consideration for the deployment of our device, we attempted to develop a minimal growth medium that could be highly concentrated in the food tanks of our final device, and fed to the cells at a low flow rate. Our results suggest that our strains could be sustained on a steady flow of 2% (w/v) sodium lactate solution. <br />
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<h6>Wet Lab</h6><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/chassis">Chassis</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly">DNA Assembly</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/arsenic">Arsenic Reporter</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/assembly/naphthalene">Naphthalene Reporter</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results">Testing &amp; Results</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/biobricks">BioBricks</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/nah_operon"><i>nah</i> Operon Expression</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/artificial_river_media">Artificial River Media</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
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<h3>Summary</h3><br />
We have developed a novel biosensing platform to be used for long-term, continuous monitoring of environmental toxins, such as arsenic and naphthalene. Previous iGEM biosensing platforms have relied on the detection of fluorescence or luminescence. We build a simple bacterium-based biosensor whereby electric current is the direct output. This offers our platforms several advantages. <br />
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In order to produce an electrical output in response to analyte, we base our biosensing solution on the well-characterized metal reduction (Mtr) pathway of Shewanella oneidensis MR-1. By shutting electrons through the Mtr pathway, MR-1 is capable of transferring electrons to inorganic solids and generating current at solid-state electrodes. In particular, we choose to utilize MtrB in our biosensing system, as it plays an essential role in localization of components in the Mtr pathway [3]. <br />
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We have designed our biosensing platform to upregulate MtrB production in the presence of analyte. To construct reporter systems for both arsenic and naphthalene, we rely on a complementation strategy based on an mtrB deficient strain of S. oneidensis MR-1 [Strain JG700 [&Delta;mtrB], 2]. The endogenous copy of mtrB has been removed in JG700; therefore, by reintroducing mtrB to this knockout strain under the control of an analyte-sensitive regulation system, we restore the functionality of mtrB in proportion to the amount of analyte present in culture media. Because MtrB is essential for electrode reduction in microbial fuel cells, we will observe a current increase in response to analyte when our engineered strains are used to inoculate bioelectrochemical reactors [4]. <br />
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Because of their capability to directly transfer electrons to acceptors outside the cell, Shewanella strains are often used in microbial electrochemical systems wherein an electrode serves as a terminal electron acceptor in the Mtr pathway. In general, a microbial electrochemical system is just like any other electrochemical cell, except that a microbe is responsible for catalyzing the oxidation/reduction reaction at either the anode or the cathode. For our purposes, we are interested in half-microbial electrochemical systems with three-electrode setups, since such systems can be easily maintained at constant conditions over extended periods of time by poising the potential of a working electrode—to which the bacteria respire—with respect to a reference.<br />
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<h3>References</h3><br />
1. Dowdesw, L., Dillon, P., Miall, A., &amp; Smol, J. P. (2010). A foundation for the future: building an environmental monitoring system for the oil sands, Environment Canada.<br />
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2. Coursolle, D., and Gralnick, J.A. (2012). Reconstruction of extracellular respiratory pathways for iron(III) reduction in Shewanella oneidensis strain MR-1. Frontiers in Microbiology 3(56)<br />
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3. Hartshorne, R. S., Reardon, C. L., Ross, D., Nuester, J., Clarke, T. A., Gates, A. J., Mills, P. C., et al. (2009). Characterization of an electron conduit between bacteria and the extracellular environment . Proceedings of the National Academy of Sciences . doi:10.1073/pnas.0900086106<br />
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4. Coursolle, D., Baron, D.B., Bond, D.R., and Gralnick, J.A. (2010). The Mtr respiratory pathway is essential for reducing flavins and electrodes in Shewanella oneidensis. Journal of Bacteriology 192(2): 467-474<br />
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5. Nivens, D.E., McKnight, T.E., Moser, S.A., Osbourn, S.J., Simpson, M.L., &amp; Sayler, G. S. (2004). Bioluminescent bioreporter integrated circuits: potentially small, rugged and inexpensive whole-cell biosensors for remote environmental monitoring. Journal of Applied Microbiology, 96(1): 33-46.<br />
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6. Siegfried, K., Endes, C., Bhuiyan, A. F. M. K., Kuppardt, A., Mattusch, J., van der Meer, J. R., Chatzinotas, A., et al. (2012). Field testing of arsenic in groundwater samples of Bangladesh using a test kit based on lyophilized bioreporter bacteria. Environmental Science &amp; Technology 46(6), 3281-7<br />
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<h6 style="margin-top:32px;">Human Practices</h6><br />
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<a href="https://2012.igem.org/Team:Cornell/project/hprac/CEA">Comprehensive Environmental Assessment</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/hprac/bioethics">Bioethics</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/hprac/oil_sands">Oil Sands</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/hprac/safety">Safety</a><br />
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<h2 class="centered">Safety Overview</h2><br />
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<h3>Safe Practices in the Lab</h3><br />
Our team conducted our work on the Cornell University campus, in the Biomedical Engineering Instructional Lab in Weill Hall, as well as in the <a href="http://angenent.bee.cornell.edu/molecularlab.html" target="_blank">Angenent Lab</a> in the Department of Biological &amp; Environmental Engineering in Riley-Robb Hall. All work was conducted in a biosafety level (BSL) 1 laboratory: this means that the strains we are using (<i>Escherichia coli</i> DH5a and WM3064, <i>Shewanella oneidensis</i> MR-1 and JG700, and <i>Pseudomonas putida</i>) are non-pathogenic and well-characterized. Before gaining access to the lab space, team members were required to:<br />
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<ol><br />
<li>complete chemical waste disposal training,</li> <br />
<li>attend an orientation with Todd Pfeiffer, the Weill Hall Facilities Director, regarding the Cornell Environmental Health &amp; Safety guidelines, and </li><br />
<li>receive specific training from <a href="http://www.bme.cornell.edu/people/adj-profile.cfm?netid=sda4"> Dr. Shivaun Archer</a>, the manager of the BME Instructional Lab, for the equipment in the lab.</li><br />
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Our standard lab practices were in compliance with the World Health Organization’s Biosafety Level 1 <a href="http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf" target="_blank">guidelines</a>. Many of our basic lab practices are described below. <br />
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<li>Researchers were required to wear gloves while in the lab space, and to remove one glove when going into lab common areas. </li><br />
<li>No gloves were allowed to leave the lab space, and no food or drink was allowed into the lab space. </li><br />
<li>Within Weill Hall, microbiological samples were transported only via the internal service elevator, to avoid contamination of public areas.</li> <br />
<li>Any materials transported between the two on-campus lab spaces were held in secondary containment during transport. </li><br />
<li>All flammable liquids were kept in a flammable storage cabinet.</li><br />
<li>The lab contained distinct waste containers for general waste, biohazard waste, biohazard sharps, and non-biohazard broken glassware.</li><br />
<li>Liquid bacterial waste was treated with bleach before being discharged into sanitary sewage.</li><br />
<li>Benchtops were decontaminated with ethanol before and after lab work was conducted. Tools that came into contact with bacteria were soaked in ethanol and flame-treated before and after use.</li><br />
<li>An autoclave was used to decontaminate growth media, glassware, tubes, pipette tips, etc. All lab members were trained in proper autoclave use by the Weill Hall Facilities Director in order to avoid dangers to researcher safety.</li><br />
<li>An emergency shower, eyewash, and first aid kit were available withing the lab space in case of emergency.</li><br />
<li>Lab notebooks were maintained in a taped-off no-glove area.</li><br />
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Safety procedures specific to our project, in addition to best microbial practices, were followed during testing with arsenic and naphthalene. We modified EH&amp;S standard operating procedures for <a href="https://static.igem.org/mediawiki/2012/d/d7/Sodium_Arsenite_SOP.pdf" target="_blank">arsenites</a>, <a href="https://static.igem.org/mediawiki/2012/3/37/Sodium_hydrogen_arsenate_heptahydrate_SOP.pdf" target="_blank">arsenates</a>, and <a href="https://static.igem.org/mediawiki/2012/c/ca/Naphthalene_SOP.pdf" target="_blank">naphthalene</a> for our specific experimental needs. Most importantly, all work with arsenic and naphthalene was conducted by team members wearing appropriate PPE in a taped-off, designated area, and with equipment labeled as arsenic- or naphthalene-contaminated. Waste was collected separately, labeled appropriately as a cancer-hazard and disposed of through the Cornell EH&amp;S.<br />
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Similar precautions were taken in working with Ethidium Bromide - all EtBr-staining was done in designated boxes, and in taped off areas used only for running and staining gels. Gels were visualized on a UV light box protected from EtBr contamination using saran wrap, which could be easily disposed of in the biohazard bin rather than risking ineffective decontamination of the UV light box. Team members were protected from exposure to UV rays with a UV shield. Facial shields were also available for use when needed.<br />
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Material Safety Data Sheets for these three hazardous compounds, as well as all other hazardous compounds used in the lab, were readily available in a binder in the lab space, as well as in the team’s shared online file folder.<br />
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<h5>Is there a local biosafety group, committee, or review board at your institution?</h5><br />
Yes, <a href="http://sp.ehs.cornell.edu/Pages/Home.aspx" target="_blank">Cornell University Environmental Health &amp; Safety</a> oversees safe procedures in on-campus laboratory research. The Cornell EH&amp;S mandates training required of researchers before they can gain access to lab spaces. Through the EH&amp;S, all team members took a required course on Chemical Waste Disposal.<br />
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<h5>If yes, what does your local biosafety group think about your project?</h5><br />
We have been in contact with the EH&amp;S at our university about best practices for testing with arsenic. We have consulted them about disposal of carcinogenic wastes, as well as about how to keep researchers safe by using secondary containment and designated equipment. The EH&amp;S has expressed satisfaction that our standard operating protocols and waste disposal plan will ensure researcher safety, as well as public safety. <br />
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<h3>Safety Concerns for Our Project</h3><br />
We are working with biosafety level 1 organisms - <i>Escherichia coli</i> DH5α and WM3064 (a conjugation-enabled DAP auxotroph), <i>Shewanella oneidensis</i> MR-1 and JG700 (an mtrB knockout strain), and <i>Pseudomonas putida</i>. None of these strains pose a threat to researcher or public health before modification. The pathways that we are modifying in <i>S. oneidensis</i> JG700 are natural pathways already found in <i>Shewanella</i> and <i>Pseudomonas</i>, so they do not raise any safety concerns. As our project aims to detect the unwanted presence of toxic compounds in the environment, our device could not easily be used to negatively impact the public or the environment, and thus does not pose any foreseeable biosecurity risk. <br />
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<h5>Would any of your project ideas raise safety issues in terms of:</h5><br />
<h6>researcher safety,</h6><br />
As we were working at BSL 1, the primary concern in terms of researcher safety was testing with our toxins of interest. Please see safe lab practices and standard operating procedures for arsenic and naphthalene, above.<br />
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<h6>public safety,</h6><br />
Again, our project itself could not be easily used to negatively impact the public. However, even when a research project does not pose a security threat, the conducting of research too must be done in a way that protects public interests and safety. All bacteria and waste were disposed of properly, in biohazard bags and through the Cornell EH&amp;S, rather than in common garbage cans, or decontaminated with bleach before being poured down the drain.<br />
<br>Additionally, in order to make our biosensor field-deployable, i.e. suitable for placement in water that will be drinking water, our device includes a thorough effluent filtration system to prevent the escape of our modified strains into the environment. We’re using a safe species and safe modifications that would not pose any foreseeable threat to native species or biodiversity. Finally, though our current selective pressure on our engineered strains is antibiotic resistance, the final device would use strains that were auxotrophs for some essential nutrient or that have our genetic modifications integrated into the bacterial chromosome; this would avoid the possibility of conferring antibiotic resistance to harmful bacterial strains in water. In the case of auxotrophic selection, our strain would be mutated such that it is an auxotroph for two essential nutrients: one of these nutrients would be provided in the media, so that the strain could only survive inside our bioreactors. Selective pressure for the plasmid would then be maintained through the second essential nutrient, which the plasmid would enable the bacteria to synthesize.<br />
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<h6>or environmental safety?</h6><br />
We do not foresee any adverse effects of deploying our device in natural streams and lakes. As detailed above, we have thought carefully about how to prevent our modified strains from getting into the environment. However, because we are knocking out mtrB - an essential component of <i>S. oneidensis</i>’s respiration pathway - and putting its production under the control of an inducible promotor, it is likely that our modified strains would be less able to compete than the wild type organisms already in the lake. If our parts were to lose their function, through mutation or other means, the most likely outcome would be that our strains would lose their capacity for facultative anaerobic respiration entirely, again leaving them less able to compete with wild-type <i>Shewanella</i>. There is the concern that antibiotic resistance could be horizontally transferred to other organisms in the wild, if our strain were to be released into the wild. This could again be addressed by selecting for strains auxotrophically.<br />
<br><br><br />
<h5>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</h5> <br />
The only safety issue associated with our new BioBrick parts is researcher safety in testing the parts. We have documented our standard operating procedures above, which other teams can reference if they want to use our parts, or test other parts using arsenic or naphthalene.<br />
<br><br><br />
We are also conducting extensive tests to assess the functionality of our BioBrick parts, to ensure that they behave as expected under different conditions.<br />
<br><br><br />
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<h3>Safety Concerns for iGEM Competition</h3><br />
<h5>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</h5> <br />
While our team is fortunate enough to have a biosafety group readily available for on-campus consultation, we recognize that many other teams may not have comparable biosafety resources available to them. In addition to the documentation by teams of their own safe practices, which can be used by other teams as references, more explicit safety guidelines, a “safety checklist” for instance, could be provided by the iGEM competition itself. One possibility is a checklist that rates a given lab from “unsafe” to “very safe” based on the number of safety provisions satisfied by the user filling out the checklist - rather like the Internet sites which assess the strength of inputted passwords.<br />
<br><br><br />
Many of the safety concerns we expressed above are the same concerns that many other iGEM teams also face. Mainly, the prominent use of antibiotic resistance in the construction and retention, via selective pressure, of novel genetic circuits poses a continued risk to public health due to the possibility of conferred antibiotic resistance. While the genes of antibiotic resistance can be carefully controlled in the lab setting, for any device to be used in a real-world setting, this possibility of horizontally transferring antibiotic resistance must be addressed. With this in mind, future iGEM teams should make an effort, where possible, to transition to other forms of selective pressures, such as limited growth media.<br />
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<h6 style="margin-top:32px;">Human Practices</h6><br />
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<a href="https://2012.igem.org/Team:Cornell/project/hprac/CEA">Comprehensive Environmental Assessment</a><br />
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<a href="https://2012.igem.org/Team:Cornell/project/hprac/bioethics">Bioethics</a><br />
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<h2 class="centered">Comprehensive Environmental Assessment</h2><br />
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<h3>Introduction to CEA</h3><br />
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As scientists, we are often inclined to reduce complex procedures down to simple, step-by-step protocols. Assessing risk and evaluating environmental impact are no exception; our first instinct this year was to create a universal checklist of regulations that every environmental iGEM project could fulfill in order to ensure environmental safety, the idea being that we could easily and systematically find answers to questions of environmental safety in scientific literature. However, upon speaking with Dr. Christina Powers, a biologist at the US Environmental Protection Agency, and further exploring the concerns relating to our own project, we decided to adopt a new approach to issues of human practices: Comprehensive Environmental Assessment (CEA).<br />
</br><br />
<br>CEA differs from traditional methods of risk assessment by recognizing that risk assessment is fundamentally a decision-making process in which scientists, experts, and the public should be engaged. The goal is to foster transparent discussion and use collective judgment to evaluate limitations and trade-offs in order to arrive at holistic conclusions about the primary issues that researchers should address in their research planning.</br><br />
<img src="https://static.igem.org/mediawiki/2012/9/97/CEA_schematic.png" class="inline"><br />
<i>Taken from http://www.synbioproject.org/process/assets/files/6609/_draft/background_packet_for_cea_and_syn_bio_workshop_jul_28_2011.pdf</i><br />
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<h3>CEA &amp; Synthetic Biology</h3><br />
While the Environmental Protection Agency primarily uses the CEA approach for nanomaterials, the Woodrow Wilson International Center for Scholars in Washington, D.C., recently launched efforts to lay out a framework to apply CEA to synthetic biology. This groundbreaking project set out to assess the CEA approach’s relevance to synthetic biology, in anticipation of the growing demand for synthetic biology-based solutions to global issues. They arrived at the conclusion that scientists should focus on four major areas of risk assessment: altered physiology, competition and biodiversity, evolutionary prediction, and gene transfer.<br />
</br><br />
<br>The Woodrow Wilson Center’s Synthetic Biology Project recommended that CEA be applied to more developed projects that were approaching field deployment in order to evaluate it as a risk-assessment approach for synthetic biology at large. This is where we come in: can CEA be successfully used to evaluate the risks of our field-deployable device? What are its limitations? </br><br />
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<br>We began by attempting to apply the Synthetic Biology Project’s modified guidelines for prioritizing research questions to our own project as it currently stands.</br><br />
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<h5>Altered Physiology</h5><br />
<br />
The current versions of our genetically modified strains have a membrane protein in the Mtr pathway for anaerobic respiration knocked out and complemented on a plasmid. They differ from wild-type <i>Shewanella oneidensis</i> MR-1 in that their capacity for anaerobic respiration is upregulated in the presence of analyte (arsenic or naphthalene). This plasmid appears to be producing mtrB in a fully functional form that successfully completes the anaerobic respiration pathway when induced. One important difference is that when uninduced, the bacteria loses much of its anaerobic respiratory activity, and must rely significantly more on aerobic respiration; this is further discussed below.<br />
<br />
<br>In addition, our salicylate reporter strain contains the nah operon, which allows the cells to constitutively degrade naphthalene into salicylate. Salicylate, a metabolic intermediate in the <i>Pseudomonas</i> strain from which the nah operon was taken, is less toxic to the environment than naphthalene. <i>P. putida</i> G7 can further degrade salicylate into a catechol, an edible carbon source using the sal operon; this second operon is not present in our cells, so our <i>Shewanella</i> strains are unable to actively degrade salicylate. This means that our cells cannot use naphthalene as a carbon source, a conclusion that is supported by our naphthalene growth assays. The effects of salicylate production on the cells appear to be negligible, but this should be further explored in future characterization work. </br><br />
<br />
<br>One of our future plans is to integrate our constructs into the chromosomes of our strains, in order to ensure that mtrB is not saturating at its uninduced, basal expression levels. This poses new questions: will chromosomal integration alter the expression patterns of this engineered pathway? Will it interfere with other functions of the cells? Functionality can be altered depending on the position of the construct within the chromosome. This is our first forthcoming research priority: determining any potential adverse effects of chromosomal integration, carrying it out, and thoroughly testing the functionality of our new engineered strains. </br><br />
<br />
<h5>Competition &amp; Biodiversity </h5><br />
A second concern is that engineered strains could possibly outcompete wild-type species in the environment and pose a threat to biodiversity. Our first line of defense against this possibility is physical containment: the inlet and outlet of our current prototype are equipped with 0.1 µm filters, effectively eliminating the possibility that our engineered strains may interact with natural organisms. However, imagine that there could be a mechanical error: physical breakage of the device, slight discrepancies in filter pore sizes, et cetera, that could leave a slim possibility that our strains could come into contact with natural species. What then?<br />
<br />
<br>To answer this question, we return to the altered physiology of our strains: the primary distinguishing characteristic to note is that the cells cannot carry out the wild-type levels of anaerobic respiration unless they are induced. This means that in situations where oxygen availability is limiting, and toxins are not highly prevalent, our strains are less able to compete than wild-type Shewanella; this is our second line of defense. <i>S. oneidensis</i> is native to freshwater ecosystems in our area, so our strains should not be disruptive to biodiversity. In addition, we have observed that inserting the nah operon into cells seems to significantly increase their doubling time, so our salicylate reporter strains would be even less able to compete than our arsenic reporters.</br><br />
<br />
<br>However, what happens in the case that oxygen availability is not limiting? It is probable that our current engineered strains would be able to survive and thrive in aerobic conditions, and live alongside wild-type <i>Shewanella</i>. We need a third line of defense for this situation, a modification that would prevent our strains from being able to survive outside of our physical device. For this, we propose auxotrophy: in a truly field-deployable device, we would use a strain that had a gene for a key metabolite knocked out, so that the strain would rely upon supplementation of this nutrient in order to survive. We would then supplement this metabolite inside of the device, so that our strains would function properly within our device. If the bacteria were to somehow escape, their functionality would be severely impaired by the lack of nutritional supplementation, and they would be quickly outcompeted by wild-type strains.</br><br />
<br />
<br>This brings to the table a second research priority for field-deployability: engineer an auxotrophic strain of <i>Shewanella</i> to contain our constructs, and ensure that the likelihood of survival without nutritional supplementation is very, very low. We would also need to ensure, from a practical standpoint, that auxotrophy would not interfere with any other aspects of the physiology of the cells.<br><br />
<br />
<br><br />
<h5>Evolutionary Prediction</h5> <br />
Due to the relatively simple nature of our reporter system, we find it highly unlikely that our strains could evolve to possess any dangerous function if somehow released into the wild. However, it is still possible that the promoter regions of our constructs could mutate and alter the expected patterns of promoter activity, or that the functionality of mtrB would be impaired by a deleterious mutation. The former possibility could serve to make the strain more genetically similar to the wild-type, and thus more able to compete; this presents us with another reason to pursue auxotrophy as a solution, basically in order to ensure that our strains are not alive for long enough for mutations to accumulate. <br />
</br><br />
<br />
<br>However, we would also need to conduct extensive testing to ensure that our strains remain functional and responsive to analyte for the full 6-month period.<br />
</br><br />
<br />
<p><br />
<h5>Gene Transfer</h5> <br />
Perhaps the most pressing concern with any synthetic biology project is the possibility of horizontal gene transfer from engineered to natural strains, and vice versa. Again, our first line of defense is physical containment, but barring that, what can we do to reduce the possibility that our strains would transfer unnatural functions to natural cells? The biggest shortcoming of our current system is that antibiotic resistance is used as a selective marker on our plasmids, thus allowing for the possibility that antibiotic resistance could be spread among natural populations via conjugation or passive transformation. While we could explore the possibility of using another form of auxotrophy as a selective marker on a plasmid, we believe that a more viable solution would be to eliminate the need for a selective pressure in the first place, namely by chromosomal integration. The transfer of chromosomal DNA is much less likely than the transfer of a plasmid, and, as mentioned above, chromosomal integration would solve other problems in the functionality of our reporters.<br />
</p><br />
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<h3>Limitations of CEA</h3><br />
CEA allowed us to think about crucial future work for our project in order to make it suitable for field-deployability. However, in our interactions with environmental groups, public officials in water quality management, and industrial groups, we encountered several important questions that were not built into existing CEA framework. Assessing the suitability of a synthetic biology-based device extends far beyond practical environmental risk assessment, into regulatory and economic concerns. The Presidential Commission for the Study of Bioethical Issues (PCSBI) released a report in December 2010 regarding important considerations for the ethics of synthetic biology projects, not all of which are encompassed by the CEA framework. In addition, as we approach field-deployability, the economics of the device become essential to assessing its viability as a solution to real-world water monitoring needs. These ideas are further explored in the remainder of this section.<br />
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<h3>References</h3><br />
Dana, G. V., Kuiken, T., Rejeski, D., &amp; Snow, A. A. (2012). Synthetic biology: Four steps to avoid a synthetic-biology disaster. <i>Nature, 483.</i> doi:10.1038/483029a</br><br />
<br>Powers, C. M., Dana, G., Gillespie, P., Gwinn, M. R., Hendren, C. O., Long, T. C., Wang, A., Davis, J. M. (2012). Comprehensive Environmental Assessment: A Meta-Assessment Approach. <i>Environ. Sci. Technol., 46,</i> 9202−9208. http://dx.doi.org/10.1021/es3023072</br><br />
<br>Synthetic Biology Project. (2011, July 28). Comprehensive Environmental Assessment and Its Application to Synthetic Biology Applications. Retrieved from http://www.synbioproject.org/events/archive/cea/</br><br />
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<h3>September 1st-8th</h3><br />
Conjugating nah into Shewy: The mysterious failure of past week's ligation lead to a redigestion (in case we run out of DNA) and religation of last week's digestions. Nah to oriT was ligated in ratios of 3:1 and 6:1 - when run on a gel, bands once more failed to appear... But wait! Epiphany! The team realized that we forgot to dephosphorylate the oriT backbone! After that silly mistake, 1:1, 3:1, and 6:1 ratios of nah:oriT were set up in a ligation mixture and electroporated into WM3064. Once again, slow growth and tiny colonies were observed - perhaps due to the strain of putting such a massive operon in a cell. 13 colonies were deemed large enough to try colony PCR, and 3 of them definitely showed bands that corresponded to the length of our construct. We set up liquid cultures for Miniprepping and once again observed slow growth. We tried subculturing - but still, general opaqueness. Miniprepping proceeded anyway, as we are a fairly optimistic bunch (and the cultures were by then over 24 hours old).<br />
<br><br><br />
Site-directed mutagenesis: After several more unsuccessful attempts, lengthy discussion among teammates, and consultation with several graduate advisors, the team decided to put this subproject on hold for the time being. Swati finished up with one last attempt, then joined Claire for fluorescence testing. That's all, folks! <a href="#" class="technical-desc" for="#technical-desc1" style="display:block;margin-top:20px;">Daily Details</a></p><br />
<div class="hide-me panel" style="background:white;margin-top: 20px;" id="technical-desc1"><br />
<h6>Daily Details:</h6><br />
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<p><strong>September 1</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Due to the mysterious failings of our attempts to ligate the nah operon, Tina tried to start fresh by re-digesting the nah operon-containing plasmid and the oriT-containing backbone.</p><br />
<br />
<p><strong>Synthetic River Media:</strong><br />
<br />
Mark and Chie created synthetic river media based on information on the mineral content of the Athabasca River, and started overnight cultures of numerous strains to test how sodium lactate supplemented river water would grow the cells.</p><br />
<br />
<p><strong>Site-directed mutagenesis:</strong> Swati transformed both her mutagenic PCR and her DpnI digest into DH5a after desalting on a membrane. She also ran both of the aforementioned samples on a gel without purifying, in order to check if either the digestion or purification steps were problematic. The gel was yet again blank, suggesting that the PCR itself was the problem.</p><br />
<br />
<p><strong>September 2nd</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Tina tried ligating an older digestion of the nah operon into the oriT backbone using a molar ratio or 3:1 and 6:1 for nah:backbone. She then de-salted and transformed the ligations into conjugation strain WM3064. Tina then ran a gel of the digested nah operon-containing plasmid and the oriT-containing backbone and extracted the nah operon and oriT backbone bands.</p><br />
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<br><br />
<b>From left: Ladder, 5 ug backbone, 5 ug backbone, 5 ug nah operon, 5 ug nah operon, 5 ug nah operon, 5 ug nah operon, empty, empty, empty:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/7/79/9_2_2012_redigest_of_oriT_and_nah_EDIT.jpg/504px-9_2_2012_redigest_of_oriT_and_nah_EDIT.jpg"><br />
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<p><strong>Synthetic River Media:</strong><br />
<br />
Mark set up a 96-well plate to test growth of the engineered strains in sodium lactate at varying concentrations, in synthetic river media.</p><br />
<br />
<p><strong>Site-directed mutagenesis:</strong>The third transformation attempt yielded no colonies on the plate of DpnI-digested DNA, but three colonies on the PCR only plate, suggesting that these were just excess template. Nonetheless, Swati set up cultures to miniprep the following day. She also realized that she had been using a template that was 10x more concentrated than it was supposed to be. She proceeded to set up a fourth attempt at a mutagenic PCR, this time with the correct template concentration, primers for mutating a different cutsite than she had previously been attempting, and a higher annealing temperature.</p><br />
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<p><strong>September 3rd</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Tina extracted the nah operon digest and the oriT backbone from yesterday's gel. Tina and Dylan had an epiphany - we forgot to dephosphorylate the backbone! No wonder we kept seeing self-ligations! With a renewed spirit, Tina dephosphorylated the digested oriT backbone then started a sixteen hour 16 degrees Celsius ligation of the extracted digests. Tina tried nah to backbone ratios of 1:1, 3:1, and 6:1.</p><br />
<br />
<p><strong>Synthetic River Media:</strong><br />
<br />
Mark returned to the lab to discover that the growth assay went wrong. Numerous issues were identified with the original protocol to explain the failed result, and the protocol was changed.</p><br />
<br />
<p><strong>Site-directed mutagenesis:</strong> Swati miniprepped the three suspicious PCR only colonies from the previous day, and digested with PstI to check for successful mutagenesis. She also did a DpnI digest and transformation of the PCR from the previous day. She ran the new PCR &amp; DpnI digest, along with the PstI-digested minipreps of the three earlier colonies, on a gel, but to no avail. The new things showed nothing (again), and the digested minipreps produced completely incorrect bands.</p><br />
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<p><strong>September 4th</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Tina desalted yesterday's ligations then transformed them into our conjugation strain WM3064. WM3064 is E. coli that is auxotrophic for DAP (diaminopimelic acid epimerase).</p><br />
<br />
<p><strong>Site-directed mutagenesis:</strong> The latest transformation did not work. Thus concludes this subproject!</p><br />
<br />
<p><strong>September 5th</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Caleb and Tina checked the transformation plates - some colonies were visible on all three ligation ratio pltaes, but the colonies were so tiny we decided to wait another day before trying colony PCRs or starting liquid cultures.</p><br />
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<p><strong>September 6th</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Caleb decided the transformation colonies were large enough to colony PCR and start liquid cultures from. 13 total colony PCRs were performed on colonies from all three ligation mixtures and one positive control was run in parallel to be sure the PCR conditions were correct. Tina ran a gel of the the colony PCRs and the positive control. As can be seen in the gel pictures, colonies 3, 4, and 8 definitely had the nah operon and many other colonies had matching middle bands. These matching middle bands likely indicate some sort of mispriming. Colony 5 was clearly the product of an oriT backbone self-ligation. Caleb and Tina started 15 mL liquid cultures of apparently nah containing colonies 3, 4, and 8 and non-nah-containing colony 5 to Miniprep from tomorrow.</p><br />
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<br><br />
<b>From top left: Ladder, colony 1, colony 2, colony 3, positive control. From bottom left: Ladder, colony 4, colony 5, colony 6, colony 7, positive control:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/f/ff/9_6_2012_colony_PCRs_2edit.jpg/503px-9_6_2012_colony_PCRs_2edit.jpg"><br />
<br><br />
<b>From left: Ladder, colony 8, colony 9, colony 10, colony 11, colony 12, colony 13, positive control:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/f/ff/9_6_2012_colony_PCRs_2edit.jpg/503px-9_6_2012_colony_PCRs_2edit.jpg"><br />
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<br />
<p><strong>Fluorescence tests:</strong> This week started late as Claire was getting over a massive cold. However, with new vim and vigor and her trusty labmate Swati, they set forth to decide the details of how they will run fluorescence tests. To get ready for this, they set up cultures of JG700 and E. coli constitutively producing mRFP under the control of Anderson series promoters.</p><br />
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<h3>September 9th-15th</h3><br />
Conjugating nah into Shewy: Unfortunately, the miniprep yields from last week were all low except one shining colony - which didn't have the nah operon in it. We ran it to test our hypothesis that slow growth of E Coli was due to the added stress on the cell of having such a large operon in it - our data so far matches our hypothesis, as that colony grew faster in culture and the plate and also yielded substantially more DNA after miniprepping.<br />
<br><br><br />
Site-directed mutagenesis: Though we were unable to confirm the presence of the nah operon with sequencing (as the miniprep failed), we decided to proceed with conjugation into Shewanella just in case the transformation succeeded. We tried to grow one of the colonies with a very strong band from last week's gel in a liquid culture, and once again, observed slow growth. We conjugated into JG700 and JG700 with SAL, a plasmid with our salicylate reporter system. Liquid cultures were made from the conjugation - unfortunately, there was no growth. We streaked new plates with the old transformation in the hopes that something would finally grow!<br />
<a href="#" class="technical-desc" for="#technical-desc7" style="display:block;margin-top:20px;">Daily Details</a><br />
<div class="hide-me panel" style="background:white;margin-top: 20px;" id="technical-desc7"><br />
<h6>Daily Details:</h6><br />
<b>September 9th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the miniprep yields today - all but colony 5 had yields below 90 ug/uL. Colony 5 had a yield of over 500 ug/uL. Our hypothesis as to why the E. coli was growing so slowly was that the E. coli was stressed from having to express the many nah operon proteins. Evidence suuporting our hypothesis includes - colony 5 had a much larger colony, grew in liquid much faster, came out as lacking the nah operon, and yielded a lot more DNA from miniprepping than the colonies that supposedly had the nah operon.<br />
<br><br><br />
<b>Synthetic River Media:</b><br />
Again, the result of the growth assay was failure. This time, it appeared that the only issue was blanking, as the negative controls were reading higher than the inoculated samples. After some discussion with Dr. Archer, a better blanking method was determined, as well as the addition of longer mixing to the protocol.<br />
<br><br><br />
<b>September 10th</b><br />
<br><br><br />
<b>Fluorescence tests: </b><br />
We dephosphorylated SAL2 digested with SpeI and PstI for one hour, then heat killed at 65degC for 10min. We then ligated overnight with mRFP (also digested with SpeI and PstI) at 16degC in the thermocycler.<br />
However, JG700 and p41k didn’t grow, so we moved the plates to Riley Robb in the hopes that a more controlled incubator meant for Shewenella will help them thrive.<br />
<br><br><br />
<b>September 11th</b><br />
<br><br><br />
<b>Fluorescence tests:</b><br />
Swati transformed 2uL of SAL2_mRFP ligation mixture into WM3064. We also made cultures of JG700, WM3064, and p39k, p40k, and p41k in both JG700 and WM3064. We are hoping to run a plate with both strains on it to get an idea of whether we can see mRFP constitutively produced in JG700 (which is naturally red and may have some background signal), and compare that fluorescence to constitutively produced mRFP in WM3064. Once we have this control data we hope to be able to correlate fluorescence in JG700 with promoter strength.<br />
<br><br><br />
<b>September 12th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb and Tina started cultures of colony #3 from original transformation plate and cultures of JG700 + SAL and JG700. JG700 is Shewanella oneidensis with a ΔmtrB genotype. SAL refers to a plasmid with our reporter system that responds to the presence of salicylate by upregulating expression of mtrB.<br />
<br><br><br />
<b>Fluorescence tests:</b><br />
Cultures of JG700 p39k and p41k didn’t grow. We made new cultures with giant swabs of cells from the plates to see if the plates are dead, and subcultured from all the other cultures started yesterday.<br />
There are also two colonies on our SAL2_mRFP transformation plate, so we made 25mL cultures from these to see if we ligated successfully.<br />
<br><br><br />
<b>September 13th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb started liquid cultures of colonies #3 and #4 from the original transformation plate. Caleb also started a conjugation of JG700 + SAL with colony #3 and JG700 + colony #3. The conjugation was at room temperature on a DAP plate for 16.5 hours.<br />
<br><br><br />
<b>Fluorescence:</b><br />
Swati miniprepped the SAL2_mRFP cultures, and Claire set up a PCR to confirm whether SAL2_RFP was ligated successfully. Unfortunately it doesn’t look like the ligations worked, as neither band is ~4.5kb, which should be the length for SAL2 with mRFP. We think that the old digestions we are using may not be good, so we will start from scratch next time.<br />
<br><br><br />
<b>September 14th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures from yesterday and noted there was no growth, as expected, due to the nah operon stressing the cells. Caleb streaked new plates from the conjugation - the LB agar plates had kanamycin and chloramphenicol for the JG700 + SAL and colony #3 conjugation and just chloramphenicol for the JG700 and colony #3 conjugation. The plasmid in the SAL strain confers kanamycin resistance and the plasmid in colony #3 with the nah operon confers chloramphenicol resistance. The strain of E. coli in colony #3 was auxotrophic for DAP, so the E. coli couldn't grow on the newly streaked plates.<br />
<br><br />
<b>From left: Ladder, SAL2_mRFP (1), SAL2_mRFP (2), mut2 pst1, mut2 miniprep:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d4/2012_09_14_sal2RFP_mut2pst1.jpg/800px-2012_09_14_sal2RFP_mut2pst1.jpg"><br />
<br />
<br><br><br />
<b>Synthetic River Media:</b><br />
Cultures of only JG700 and Sal1 containing cells were started today, for another attempt at the growth assay.<br />
<br><br><br />
<b>September 15th</b><br />
<br><br><br />
<b>Running Reactors:</b><br />
Because maximal current production did not increase, Dylan took down reactors with working electrodes poised at 0.35V with respect the the Ag/AgCl reference electrode in order to free up potentiostat channels so that we can begin characterizing our arsenic reporter strains.<br />
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<h3>September 16th-22nd</h3><br />
Conjugating nah into Shewy: Some colonies were observed from last week's restreaking! A massive 15 colony liquid culture was set up, but to no avail. After five long days, we were forced to conclude that conjugation failed. Sadface. Good thing we had extra 3:1 nah to oriT ligation sitting around, which we transformed into both WM3064 and DH5a, the latter of which we hoped would grow faster. 16 colonies from the Sep 4th ligation were also restreaked. And lo! Every single restreak had colonies the next day, which warranted a massive 16 colony colony PCR (try saying that five times fast). Interestingly, the WM3064 plates grew, but the DH5a transformation failed, which we concluded was due to a bad electrocompetent cell stock (we noticed weird white precipitate in the cell mixture before).<br />
<a href="#" class="technical-desc" for="#technical-desc2" style="display:block;margin-top:20px;">Daily Details</a><br />
<div class="hide-me panel" style="background:white;margin-top: 20px;" id="technical-desc2"><br />
<h6>Daily Details:</h6><br />
<b>September 16th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the streaked conjugation plates - there were some colonies, but they were all on top of the initial streak. He and Tina started liquid cultures of all 15 colonies.<br />
<br><br><br />
<b>Synthetic River Media:</b><br />
After finalizing the growth assay protocol, another experiment was set up, this time with only two strains (JG700 and Sal1) in triplicate with sodium lactate concentrations (varying from none to full M4 media concentrations) in synthetic Athabasca river water, plus positive LB controls and blanks. The assay was set to run at room temperature for 20 hours, taking data every 5 minutes with 30 seconds of mixing before each read. Blanking was done by group, for each media mixture.<br />
<br><br><br />
<b>Fluorescence tests:</b><br />
Started liquid cultures of JG700, p25a, and p29a in order to re-conjugate these constitutively produced mRFP plasmids into Shewanella. p25a on a pBBRBB backbone in WM3064 corresponds to p39k in JG700, and p29a corresponds to p41k.<br />
<br><br><br />
<br />
<b>September 17th</b><br />
<br><br><br />
<b>Running Reactors:</b> After autoclaving a reactor setup and getting peristaltic pumps ready for a new continuous flow experiment, Dylan started a liquid culture of an arsenic reporter strain (JG700 + p14k) to be inoculated into reactors.<br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started yesterday - no growth.<br />
<br><br><br />
<b>Synthetic River Media:</b><br />
The growth assay was successful, with constant and low OD600 in the blank wells, and a standard growth curve in the LB wells. The data suggests that lowering the sodium lactate from M4 concentrations is impossible. Therefore, a solution containing 5% volume/volume of 40% sodium lactate weight/volume is what is necessary to maintain an OD600 of approximately 0.1 for an extended period of time. However, the supported OD600 may be higher in the final device at this concentration, due to the constant flow of new sodium lactate.<br />
<br><br><br />
<b>Fluorescence tests:</b><br />
We redigested SAL2 and mRFP with SpeI and PstI-HF to try to clone mRFP into our salicylate reporter, so that we can do fluorescence experiments with the two salicylate parts. Swati also plated WM3064 with JG700 for conjugation of the constitutively produced mRFP.<br />
<br><br><br />
<b>September 18th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started on the 16th - no growth.<br />
Fluorescence tests: We ran mRFP on a gel and extracted a 800bp band which was the appropriate link for mRFP. After enzyme purifying the SAL2 digestion and dephosphorylating, Claire set up an overnight ligation of SAL2 and mRFP.<br />
<br><br><br />
<b>September 19th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started on the 16th - no growth.<br />
Fluorescence tests: Claire transformed the SAL2_mRFP ligation into WM3064, and set up cultures of p39k and p41k from the conjugation plate.<br />
<br><br><br />
<b>September 20th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started on the 16th - no growth.<br />
Fluorescence test: Swati miniprepped cultures of p39k and p41k from the conjugation plates so that we can PCR to confirm if we have successfully conjugated constitutive mRFP into Shewanella.<br />
<br><br><br />
<b>September 21st</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started on the 16th - no growth. He concluded that the conjugation failed. Caleb and Tina electroporated the 3:1 (nah:oriT backbone) ligation of dephosphorylated and digested nah with the oriT backbone from September 3rd into both WM3064 and a DH5α strain. They then streaked 16 colonies from the September 4th transformation onto fresh plates.<br />
<br><br><br />
<b>September 22nd</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb and Tina noted the appearance of the re-streaked plates - every single streak had numerous colonies. They started liquid cultures and started an overnight colony PCR of all 16 of them. Included with the colony PCRs was a positive control that we knew would have the same amplicon from the nah operon if the colonies were the result of a successful ligation. The electroporated WM3064 plate grew with numerous isolated colonies; the DH5α transformation failed. We hypothesized the DH5α cells were at fault due to their appearance before electroporation - white precipitate was floating in the cell mixture.<br />
<br><br><br />
<b>Fluorescence tests:</b> Using samples from the 20th, Swati did a Phusion PCR to see if we successfully conjugated p39k and p41k into Shewanella. She also miniprepped WM3064 SAL2RFP and did PCR to see if that ligation was successful.<br />
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<h3>September 23rd-30th</h3><br />
Conjugating nah into Shewy: A gel of last week's colony PCRs hinted that one of the colonies had the nah operon in it! Excitement! Liquid cultures were made, and there was just enough DNA after miniprepping to submit for sequencing. We will collectively hold our breaths and wait for positive results.<br><br><br />
This week, we were able to begin characterizing the current response to arsenic of S20 (JG700 + p14k; see strain list). Additionally, we submitted physical DNA for six BioBrick parts to the parts registry.<br />
<a href="#" class="technical-desc" for="#technical-desc3" style="display:block;margin-top:20px;">Daily Details</a><br />
<div class="hide-me panel" style="background:white;margin-top: 20px;" id="technical-desc3"><br />
<h6>Daily Details:</h6><br />
<b>September 23rd</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb and Tina ran a gel of the colony PCRs and positive control PCR. Swati also started cultures of our arsenic-sensitive strain, as well as control strains, for a plate testing our arsenic reporters.<br />
<br><br />
<b>From top-left: ladder, positive control, 8 test colonies. From bottom-left: ladder, 8 test colonies, and Swati's SAL2 RFP:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/2012_9_23_another_colony_PCR.jpg/703px-2012_9_23_another_colony_PCR.jpg"><br />
<br><br />
<b>From left: ladder, empty, p39k PCR, p41k PCR, empty, SAL2_mRFP PCR:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/ef/2012_09_23_FloFloFlo.jpg/496px-2012_09_23_FloFloFlo.jpg"><br />
<br />
<br />
<br><br><br />
<b>September 24th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb inoculated 30 mL LB+DAP+Cm with the 24 hour liquid culture of of colony N.<br />
<br><br><br />
<b>September 25th</b><br />
<br><br><br />
<b>Fluorescence tests:</b> Swati set up a conjugation of SAL2_mRFP into JG700. Also, sequencing came back good so once we have this part in Shewanella we can start testing our salicylate sensor. Jim and Swati also set up a plate to test our arsenic sensing parts at different concentrations of arsenic – they ran a plate with a blank LB column, five control columns (JG700, MR-1, p39k, p40k, and p41k), and three columns of each of our arsenic-sensitive strains. To each row they added a different concentration of arsenic, going from 0uM to 5uM arsenic. The final OD of 100uL in each well was 0.8, and the plate was left overnight in the plate reader.<br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb miniprepped the 36 hour 30 mL culture of colony N. The size of the pellet after the first spin step was similar to the size of a ~5mL normal culture, consequently, the yield was only 96.7 ng/uL.<br />
<br><br><br />
<b>September 27th</b><br />
<br><br><br />
<b>Running Reactors:</b> For the first time, we added arsenic to our reactors. Specifically, once Dylan saw that the two reactors inoculated with an arsenic reporter strain (JG700+p14k) had been producing steady current for several retention times, he dosed the media vessel to a final concentration of 10 μM sodium arsenite.<br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Fortunately, there was enough DNA for Caleb to submit the miniprepped DNA from colony N for Sanger sequencing today. Tina started 3 x (1mL cultures of colony N).<br />
<br><br><br />
<b>Fluorescence tests:</b> Claire and Swati set up another control plate with constitutively produced mRFP in both WM3064 and JG700. Since using 100uL starting at and OD of 0.8 seemed to work well for the arsenic plate, we set up the control plate with the same parameters and left it in the plate reader overnight.<br />
<br><br><br />
<b>September 28th</b><br />
<br><br><br />
<b>Running Reactors:</b> Since several retention times passed without any current response to 10 μM sodium arsenite, Dylan increased the concentration in the media vessel, hitting our reactors with 100 μM arsenite.<br />
<br><br><br />
<b>Fluorescence tests:</b> We noticed that the arsenic data did not seem to have any signal above background, and realized that it may have been because the plate reader wasn’t set to read a clear-bottomed plate. We concluded that, since from the test done on the 27th we could see distinct difference between fluorescence levels of constitutively produced mRFP, our experimental procedure is good and the plate reader was just on the wrong setting. Additionally, since the bioreactors are not getting upregulation of arsenic up to 10uM, we decided next time to test higher concentrations of arsenic (0uM to 500uM)<br />
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<h3>September 1st-8th</h3><br />
<p><strong>September 1</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Due to the mysterious failings of our attempts to ligate the nah operon, Tina tried to start fresh by re-digesting the nah operon-containing plasmid and the oriT-containing backbone.</p><br />
<br />
<p><strong>Synthetic River Media:</strong><br />
<br />
Mark and Chie created synthetic river media based on information on the mineral content of the Athabasca River, and started overnight cultures of numerous strains to test how sodium lactate supplemented river water would grow the cells.</p><br />
<br />
<p><strong>Site-directed mutagenesis:</strong> Swati transformed both her mutagenic PCR and her DpnI digest into DH5a after desalting on a membrane. She also ran both of the aforementioned samples on a gel without purifying, in order to check if either the digestion or purification steps were problematic. The gel was yet again blank, suggesting that the PCR itself was the problem.</p><br />
<br />
<p><strong>September 2nd</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Tina tried ligating an older digestion of the nah operon into the oriT backbone using a molar ratio or 3:1 and 6:1 for nah:backbone. She then de-salted and transformed the ligations into conjugation strain WM3064. Tina then ran a gel of the digested nah operon-containing plasmid and the oriT-containing backbone and extracted the nah operon and oriT backbone bands.</p><br />
<br><br />
<b>From left: Ladder, 5 ug backbone, 5 ug backbone, 5 ug nah operon, 5 ug nah operon, 5 ug nah operon, 5 ug nah operon, empty, empty, empty:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/7/79/9_2_2012_redigest_of_oriT_and_nah_EDIT.jpg/504px-9_2_2012_redigest_of_oriT_and_nah_EDIT.jpg"><br />
<br />
<br />
<p><strong>Synthetic River Media:</strong><br />
<br />
Mark set up a 96-well plate to test growth of the engineered strains in sodium lactate at varying concentrations, in synthetic river media.</p><br />
<br />
<p><strong>Site-directed mutagenesis:</strong>The third transformation attempt yielded no colonies on the plate of DpnI-digested DNA, but three colonies on the PCR only plate, suggesting that these were just excess template. Nonetheless, Swati set up cultures to miniprep the following day. She also realized that she had been using a template that was 10x more concentrated than it was supposed to be. She proceeded to set up a fourth attempt at a mutagenic PCR, this time with the correct template concentration, primers for mutating a different cutsite than she had previously been attempting, and a higher annealing temperature.</p><br />
<br />
<p><strong>September 3rd</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Tina extracted the nah operon digest and the oriT backbone from yesterday's gel. Tina and Dylan had an epiphany - we forgot to dephosphorylate the backbone! No wonder we kept seeing self-ligations! With a renewed spirit, Tina dephosphorylated the digested oriT backbone then started a sixteen hour 16 degrees Celsius ligation of the extracted digests. Tina tried nah to backbone ratios of 1:1, 3:1, and 6:1.</p><br />
<br />
<p><strong>Synthetic River Media:</strong><br />
<br />
Mark returned to the lab to discover that the growth assay went wrong. Numerous issues were identified with the original protocol to explain the failed result, and the protocol was changed.</p><br />
<br />
<p><strong>Site-directed mutagenesis:</strong> Swati miniprepped the three suspicious PCR only colonies from the previous day, and digested with PstI to check for successful mutagenesis. She also did a DpnI digest and transformation of the PCR from the previous day. She ran the new PCR &amp; DpnI digest, along with the PstI-digested minipreps of the three earlier colonies, on a gel, but to no avail. The new things showed nothing (again), and the digested minipreps produced completely incorrect bands.</p><br />
<br />
<p><strong>September 4th</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Tina desalted yesterday's ligations then transformed them into our conjugation strain WM3064. WM3064 is E. coli that is auxotrophic for DAP (diaminopimelic acid epimerase).</p><br />
<br />
<p><strong>Site-directed mutagenesis:</strong> The latest transformation did not work. Thus concludes this subproject!</p><br />
<br />
<p><strong>September 5th</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Caleb and Tina checked the transformation plates - some colonies were visible on all three ligation ratio pltaes, but the colonies were so tiny we decided to wait another day before trying colony PCRs or starting liquid cultures.</p><br />
<br />
<p><strong>September 6th</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Caleb decided the transformation colonies were large enough to colony PCR and start liquid cultures from. 13 total colony PCRs were performed on colonies from all three ligation mixtures and one positive control was run in parallel to be sure the PCR conditions were correct. Tina ran a gel of the the colony PCRs and the positive control. As can be seen in the gel pictures, colonies 3, 4, and 8 definitely had the nah operon and many other colonies had matching middle bands. These matching middle bands likely indicate some sort of mispriming. Colony 5 was clearly the product of an oriT backbone self-ligation. Caleb and Tina started 15 mL liquid cultures of apparently nah containing colonies 3, 4, and 8 and non-nah-containing colony 5 to Miniprep from tomorrow.</p><br />
<br><br />
<b>From top left: Ladder, colony 1, colony 2, colony 3, positive control. From bottom left: Ladder, colony 4, colony 5, colony 6, colony 7, positive control:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/f/ff/9_6_2012_colony_PCRs_2edit.jpg/503px-9_6_2012_colony_PCRs_2edit.jpg"><br />
<br><br />
<b>From left: Ladder, colony 8, colony 9, colony 10, colony 11, colony 12, colony 13, positive control:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/f/ff/9_6_2012_colony_PCRs_2edit.jpg/503px-9_6_2012_colony_PCRs_2edit.jpg"><br />
<br />
<p><strong>Fluorescence tests:</strong> This week started late as Claire was getting over a massive cold. However, with new vim and vigor and her trusty labmate Swati, they set forth to decide the details of how they will run fluorescence tests. To get ready for this, they set up cultures of JG700 and E. coli constitutively producing mRFP under the control of Anderson series promoters.</p><br />
<br />
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<h3>September 9th-15th</h3><br />
<b>September 9th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the miniprep yields today - all but colony 5 had yields below 90 ug/uL. Colony 5 had a yield of over 500 ug/uL. Our hypothesis as to why the E. coli was growing so slowly was that the E. coli was stressed from having to express the many nah operon proteins. Evidence suuporting our hypothesis includes - colony 5 had a much larger colony, grew in liquid much faster, came out as lacking the nah operon, and yielded a lot more DNA from miniprepping than the colonies that supposedly had the nah operon.<br />
<br><br><br />
<b>Synthetic River Media:</b><br />
Again, the result of the growth assay was failure. This time, it appeared that the only issue was blanking, as the negative controls were reading higher than the inoculated samples. After some discussion with Dr. Archer, a better blanking method was determined, as well as the addition of longer mixing to the protocol.<br />
<br><br><br />
<b>September 10th</b><br />
<br><br><br />
<b>Fluorescence tests: </b><br />
We dephosphorylated SAL2 digested with SpeI and PstI for one hour, then heat killed at 65degC for 10min. We then ligated overnight with mRFP (also digested with SpeI and PstI) at 16degC in the thermocycler.<br />
However, JG700 and p41k didn’t grow, so we moved the plates to Riley Robb in the hopes that a more controlled incubator meant for Shewenella will help them thrive.<br />
<br><br><br />
<b>September 11th</b><br />
<br><br><br />
<b>Fluorescence tests:</b><br />
Swati transformed 2uL of SAL2_mRFP ligation mixture into WM3064. We also made cultures of JG700, WM3064, and p39k, p40k, and p41k in both JG700 and WM3064. We are hoping to run a plate with both strains on it to get an idea of whether we can see mRFP constitutively produced in JG700 (which is naturally red and may have some background signal), and compare that fluorescence to constitutively produced mRFP in WM3064. Once we have this control data we hope to be able to correlate fluorescence in JG700 with promoter strength.<br />
<br><br><br />
<b>September 12th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb and Tina started cultures of colony #3 from original transformation plate and cultures of JG700 + SAL and JG700. JG700 is Shewanella oneidensis with a ΔmtrB genotype. SAL refers to a plasmid with our reporter system that responds to the presence of salicylate by upregulating expression of mtrB.<br />
<br><br><br />
<b>Fluorescence tests:</b><br />
Cultures of JG700 p39k and p41k didn’t grow. We made new cultures with giant swabs of cells from the plates to see if the plates are dead, and subcultured from all the other cultures started yesterday.<br />
There are also two colonies on our SAL2_mRFP transformation plate, so we made 25mL cultures from these to see if we ligated successfully.<br />
<br><br><br />
<b>September 13th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb started liquid cultures of colonies #3 and #4 from the original transformation plate. Caleb also started a conjugation of JG700 + SAL with colony #3 and JG700 + colony #3. The conjugation was at room temperature on a DAP plate for 16.5 hours.<br />
<br><br><br />
<b>Fluorescence:</b><br />
Swati miniprepped the SAL2_mRFP cultures, and Claire set up a PCR to confirm whether SAL2_RFP was ligated successfully. Unfortunately it doesn’t look like the ligations worked, as neither band is ~4.5kb, which should be the length for SAL2 with mRFP. We think that the old digestions we are using may not be good, so we will start from scratch next time.<br />
<br><br><br />
<b>September 14th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures from yesterday and noted there was no growth, as expected, due to the nah operon stressing the cells. Caleb streaked new plates from the conjugation - the LB agar plates had kanamycin and chloramphenicol for the JG700 + SAL and colony #3 conjugation and just chloramphenicol for the JG700 and colony #3 conjugation. The plasmid in the SAL strain confers kanamycin resistance and the plasmid in colony #3 with the nah operon confers chloramphenicol resistance. The strain of E. coli in colony #3 was auxotrophic for DAP, so the E. coli couldn't grow on the newly streaked plates.<br />
<br><br />
<b>From left: Ladder, SAL2_mRFP (1), SAL2_mRFP (2), mut2 pst1, mut2 miniprep:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d4/2012_09_14_sal2RFP_mut2pst1.jpg/800px-2012_09_14_sal2RFP_mut2pst1.jpg"><br />
<br />
<br><br><br />
<b>Synthetic River Media:</b><br />
Cultures of only JG700 and Sal1 containing cells were started today, for another attempt at the growth assay.<br />
<br><br><br />
<b>September 15th</b><br />
<br><br><br />
<b>Running Reactors:</b><br />
Because maximal current production did not increase, Dylan took down reactors with working electrodes poised at 0.35V with respect the the Ag/AgCl reference electrode in order to free up potentiostat channels so that we can begin characterizing our arsenic reporter strains.<br />
</div><br />
<div class="three columns"><br />
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<h3>September 16th-22nd</h3><br />
<b>September 16th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the streaked conjugation plates - there were some colonies, but they were all on top of the initial streak. He and Tina started liquid cultures of all 15 colonies.<br />
<br><br><br />
<b>Synthetic River Media:</b><br />
After finalizing the growth assay protocol, another experiment was set up, this time with only two strains (JG700 and Sal1) in triplicate with sodium lactate concentrations (varying from none to full M4 media concentrations) in synthetic Athabasca river water, plus positive LB controls and blanks. The assay was set to run at room temperature for 20 hours, taking data every 5 minutes with 30 seconds of mixing before each read. Blanking was done by group, for each media mixture.<br />
<br><br><br />
<b>Fluorescence tests:</b><br />
Started liquid cultures of JG700, p25a, and p29a in order to re-conjugate these constitutively produced mRFP plasmids into Shewanella. p25a on a pBBRBB backbone in WM3064 corresponds to p39k in JG700, and p29a corresponds to p41k.<br />
<br><br><br />
<br />
<b>September 17th</b><br />
<br><br><br />
<b>Running Reactors:</b> After autoclaving a reactor setup and getting peristaltic pumps ready for a new continuous flow experiment, Dylan started a liquid culture of an arsenic reporter strain (JG700 + p14k) to be inoculated into reactors.<br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started yesterday - no growth.<br />
<br><br><br />
<b>Synthetic River Media:</b><br />
The growth assay was successful, with constant and low OD600 in the blank wells, and a standard growth curve in the LB wells. The data suggests that lowering the sodium lactate from M4 concentrations is impossible. Therefore, a solution containing 5% volume/volume of 40% sodium lactate weight/volume is what is necessary to maintain an OD600 of approximately 0.1 for an extended period of time. However, the supported OD600 may be higher in the final device at this concentration, due to the constant flow of new sodium lactate.<br />
<br><br><br />
<b>Fluorescence tests:</b><br />
We redigested SAL2 and mRFP with SpeI and PstI-HF to try to clone mRFP into our salicylate reporter, so that we can do fluorescence experiments with the two salicylate parts. Swati also plated WM3064 with JG700 for conjugation of the constitutively produced mRFP.<br />
<br><br><br />
<b>September 18th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started on the 16th - no growth.<br />
Fluorescence tests: We ran mRFP on a gel and extracted a 800bp band which was the appropriate link for mRFP. After enzyme purifying the SAL2 digestion and dephosphorylating, Claire set up an overnight ligation of SAL2 and mRFP.<br />
<br><br><br />
<b>September 19th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started on the 16th - no growth.<br />
Fluorescence tests: Claire transformed the SAL2_mRFP ligation into WM3064, and set up cultures of p39k and p41k from the conjugation plate.<br />
<br><br><br />
<b>September 20th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started on the 16th - no growth.<br />
Fluorescence test: Swati miniprepped cultures of p39k and p41k from the conjugation plates so that we can PCR to confirm if we have successfully conjugated constitutive mRFP into Shewanella.<br />
<br><br><br />
<b>September 21st</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started on the 16th - no growth. He concluded that the conjugation failed. Caleb and Tina electroporated the 3:1 (nah:oriT backbone) ligation of dephosphorylated and digested nah with the oriT backbone from September 3rd into both WM3064 and a DH5α strain. They then streaked 16 colonies from the September 4th transformation onto fresh plates.<br />
<br><br><br />
<b>September 22nd</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb and Tina noted the appearance of the re-streaked plates - every single streak had numerous colonies. They started liquid cultures and started an overnight colony PCR of all 16 of them. Included with the colony PCRs was a positive control that we knew would have the same amplicon from the nah operon if the colonies were the result of a successful ligation. The electroporated WM3064 plate grew with numerous isolated colonies; the DH5α transformation failed. We hypothesized the DH5α cells were at fault due to their appearance before electroporation - white precipitate was floating in the cell mixture.<br />
<br><br><br />
<b>Fluorescence tests:</b> Using samples from the 20th, Swati did a Phusion PCR to see if we successfully conjugated p39k and p41k into Shewanella. She also miniprepped WM3064 SAL2RFP and did PCR to see if that ligation was successful.<br />
</div><br />
<div class="three columns"><br />
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<h3>September 23rd-30th</h3><br />
<b>September 23rd</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb and Tina ran a gel of the colony PCRs and positive control PCR. Swati also started cultures of our arsenic-sensitive strain, as well as control strains, for a plate testing our arsenic reporters.<br />
<br><br />
<b>From top-left: ladder, positive control, 8 test colonies. From bottom-left: ladder, 8 test colonies, and Swati's SAL2 RFP:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/2012_9_23_another_colony_PCR.jpg/703px-2012_9_23_another_colony_PCR.jpg"><br />
<br><br />
<b>From left: ladder, empty, p39k PCR, p41k PCR, empty, SAL2_mRFP PCR:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/ef/2012_09_23_FloFloFlo.jpg/496px-2012_09_23_FloFloFlo.jpg"><br />
<br />
<br><br><br />
<b>September 24th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb inoculated 30 mL LB+DAP+Cm with the 24 hour liquid culture of of colony N.<br />
<br><br><br />
<b>September 25th</b><br />
<br><br><br />
<b>Fluorescence tests:</b> Swati set up a conjugation of SAL2_mRFP into JG700. Also, sequencing came back good so once we have this part in Shewanella we can start testing our salicylate sensor. Jim and Swati also set up a plate to test our arsenic sensing parts at different concentrations of arsenic – they ran a plate with a blank LB column, five control columns (JG700, MR-1, p39k, p40k, and p41k), and three columns of each of our arsenic-sensitive strains. To each row they added a different concentration of arsenic, going from 0uM to 5uM arsenic. The final OD of 100uL in each well was 0.8, and the plate was left overnight in the plate reader.<br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb miniprepped the 36 hour 30 mL culture of colony N. The size of the pellet after the first spin step was similar to the size of a ~5mL normal culture, consequently, the yield was only 96.7 ng/uL.<br />
<br><br><br />
<b>September 27th</b><br />
<br><br><br />
<b>Running Reactors:</b> For the first time, we added arsenic to our reactors. Specifically, once Dylan saw that the two reactors inoculated with an arsenic reporter strain (JG700+p14k) had been producing steady current for several retention times, he dosed the media vessel to a final concentration of 10 μM sodium arsenite.<br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Fortunately, there was enough DNA for Caleb to submit the miniprepped DNA from colony N for Sanger sequencing today. Tina started 3 x (1mL cultures of colony N).<br />
<br><br><br />
<b>Fluorescence tests:</b> Claire and Swati set up another control plate with constitutively produced mRFP in both WM3064 and JG700. Since using 100uL starting at and OD of 0.8 seemed to work well for the arsenic plate, we set up the control plate with the same parameters and left it in the plate reader overnight.<br />
<br><br><br />
<b>September 28th</b><br />
<br><br><br />
<b>Running Reactors:</b> Since several retention times passed without any current response to 10 μM sodium arsenite, Dylan increased the concentration in the media vessel, hitting our reactors with 100 μM arsenite.<br />
<br><br><br />
<b>Fluorescence tests:</b> We noticed that the arsenic data did not seem to have any signal above background, and realized that it may have been because the plate reader wasn’t set to read a clear-bottomed plate. We concluded that, since from the test done on the 27th we could see distinct difference between fluorescence levels of constitutively produced mRFP, our experimental procedure is good and the plate reader was just on the wrong setting. Additionally, since the bioreactors are not getting upregulation of arsenic up to 10uM, we decided next time to test higher concentrations of arsenic (0uM to 500uM)<br />
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<h3>September 1st-8th</h3><br />
Conjugating nah into Shewy: The mysterious failure of past week's ligation lead to a redigestion (in case we run out of DNA) and religation of last week's digestions. Nah to oriT was ligated in ratios of 3:1 and 6:1 - when run on a gel, bands once more failed to appear... But wait! Epiphany! The team realized that we forgot to dephosphorylate the oriT backbone! After that silly mistake, 1:1, 3:1, and 6:1 ratios of nah:oriT were set up in a ligation mixture and electroporated into WM3064. Once again, slow growth and tiny colonies were observed - perhaps due to the strain of putting such a massive operon in a cell. 13 colonies were deemed large enough to try colony PCR, and 3 of them definitely showed bands that corresponded to the length of our construct. We set up liquid cultures for Miniprepping and once again observed slow growth. We tried subculturing - but still, general opaqueness. Miniprepping proceeded anyway, as we are a fairly optimistic bunch (and the cultures were by then over 24 hours old).<br />
<br><br><br />
Site-directed mutagenesis: After several more unsuccessful attempts, lengthy discussion among teammates, and consultation with several graduate advisors, the team decided to put this subproject on hold for the time being. Swati finished up with one last attempt, then joined Claire for fluorescence testing. That's all, folks! <br />
<div class="panel" style="background:white;margin-top: 20px;"><br />
<h6>Daily Details:</h6><br />
<p><strong>September 1</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Due to the mysterious failings of our attempts to ligate the nah operon, Tina tried to start fresh by re-digesting the nah operon-containing plasmid and the oriT-containing backbone.</p><br />
<br />
<p><strong>Synthetic River Media:</strong><br />
<br />
Mark and Chie created synthetic river media based on information on the mineral content of the Athabasca River, and started overnight cultures of numerous strains to test how sodium lactate supplemented river water would grow the cells.</p><br />
<br />
<p><strong>Site-directed mutagenesis:</strong> Swati transformed both her mutagenic PCR and her DpnI digest into DH5a after desalting on a membrane. She also ran both of the aforementioned samples on a gel without purifying, in order to check if either the digestion or purification steps were problematic. The gel was yet again blank, suggesting that the PCR itself was the problem.</p><br />
<br />
<p><strong>September 2nd</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Tina tried ligating an older digestion of the nah operon into the oriT backbone using a molar ratio or 3:1 and 6:1 for nah:backbone. She then de-salted and transformed the ligations into conjugation strain WM3064. Tina then ran a gel of the digested nah operon-containing plasmid and the oriT-containing backbone and extracted the nah operon and oriT backbone bands.</p><br />
<br><br />
<b>From left: Ladder, 5 ug backbone, 5 ug backbone, 5 ug nah operon, 5 ug nah operon, 5 ug nah operon, 5 ug nah operon, empty, empty, empty:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/7/79/9_2_2012_redigest_of_oriT_and_nah_EDIT.jpg/504px-9_2_2012_redigest_of_oriT_and_nah_EDIT.jpg"><br />
<br />
<br />
<p><strong>Synthetic River Media:</strong><br />
<br />
Mark set up a 96-well plate to test growth of the engineered strains in sodium lactate at varying concentrations, in synthetic river media.</p><br />
<br />
<p><strong>Site-directed mutagenesis:</strong>The third transformation attempt yielded no colonies on the plate of DpnI-digested DNA, but three colonies on the PCR only plate, suggesting that these were just excess template. Nonetheless, Swati set up cultures to miniprep the following day. She also realized that she had been using a template that was 10x more concentrated than it was supposed to be. She proceeded to set up a fourth attempt at a mutagenic PCR, this time with the correct template concentration, primers for mutating a different cutsite than she had previously been attempting, and a higher annealing temperature.</p><br />
<br />
<br />
<p><strong>September 3rd</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Tina extracted the nah operon digest and the oriT backbone from yesterday's gel. Tina and Dylan had an epiphany - we forgot to dephosphorylate the backbone! No wonder we kept seeing self-ligations! With a renewed spirit, Tina dephosphorylated the digested oriT backbone then started a sixteen hour 16 degrees Celsius ligation of the extracted digests. Tina tried nah to backbone ratios of 1:1, 3:1, and 6:1.</p><br />
<br />
<p><strong>Synthetic River Media:</strong><br />
<br />
Mark returned to the lab to discover that the growth assay went wrong. Numerous issues were identified with the original protocol to explain the failed result, and the protocol was changed.</p><br />
<br />
<p><strong>Site-directed mutagenesis:</strong> Swati miniprepped the three suspicious PCR only colonies from the previous day, and digested with PstI to check for successful mutagenesis. She also did a DpnI digest and transformation of the PCR from the previous day. She ran the new PCR &amp; DpnI digest, along with the PstI-digested minipreps of the three earlier colonies, on a gel, but to no avail. The new things showed nothing (again), and the digested minipreps produced completely incorrect bands.</p><br />
<br />
<p><strong>September 4th</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Tina desalted yesterday's ligations then transformed them into our conjugation strain WM3064. WM3064 is E. coli that is auxotrophic for DAP (diaminopimelic acid epimerase).</p><br />
<br />
<p><strong>Site-directed mutagenesis:</strong> The latest transformation did not work. Thus concludes this subproject!</p><br />
<br />
<p><strong>September 5th</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Caleb and Tina checked the transformation plates - some colonies were visible on all three ligation ratio pltaes, but the colonies were so tiny we decided to wait another day before trying colony PCRs or starting liquid cultures.</p><br />
<br />
<p><strong>September 6th</strong></p><br />
<br />
<p><strong>Conjugating nah into Shewy:</strong><br />
<br />
Caleb decided the transformation colonies were large enough to colony PCR and start liquid cultures from. 13 total colony PCRs were performed on colonies from all three ligation mixtures and one positive control was run in parallel to be sure the PCR conditions were correct. Tina ran a gel of the the colony PCRs and the positive control. As can be seen in the gel pictures, colonies 3, 4, and 8 definitely had the nah operon and many other colonies had matching middle bands. These matching middle bands likely indicate some sort of mispriming. Colony 5 was clearly the product of an oriT backbone self-ligation. Caleb and Tina started 15 mL liquid cultures of apparently nah containing colonies 3, 4, and 8 and non-nah-containing colony 5 to Miniprep from tomorrow.</p><br />
<br><br />
<b>From top left: Ladder, colony 1, colony 2, colony 3, positive control. From bottom left: Ladder, colony 4, colony 5, colony 6, colony 7, positive control:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/f/ff/9_6_2012_colony_PCRs_2edit.jpg/503px-9_6_2012_colony_PCRs_2edit.jpg"><br />
<br><br />
<b>From left: Ladder, colony 8, colony 9, colony 10, colony 11, colony 12, colony 13, positive control:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/f/ff/9_6_2012_colony_PCRs_2edit.jpg/503px-9_6_2012_colony_PCRs_2edit.jpg"><br />
<br />
<p><strong>Fluorescence tests:</strong> This week started late as Claire was getting over a massive cold. However, with new vim and vigor and her trusty labmate Swati, they set forth to decide the details of how they will run fluorescence tests. To get ready for this, they set up cultures of JG700 and E. coli constitutively producing mRFP under the control of Anderson series promoters.</p><br />
</div><br />
</div><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/2/26/Cornell12_Stock_10.jpg"><br />
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<h3>September 9th-15th</h3><br />
Conjugating nah into Shewy: Unfortunately, the miniprep yields from last week were all low except one shining colony - which didn't have the nah operon in it. We ran it to test our hypothesis that slow growth of E Coli was due to the added stress on the cell of having such a large operon in it - our data so far matches our hypothesis, as that colony grew faster in culture and the plate and also yielded substantially more DNA after miniprepping.<br />
<br><br><br />
Site-directed mutagenesis: Though we were unable to confirm the presence of the nah operon with sequencing (as the miniprep failed), we decided to proceed with conjugation into Shewanella just in case the transformation succeeded. We tried to grow one of the colonies with a very strong band from last week's gel in a liquid culture, and once again, observed slow growth. We conjugated into JG700 and JG700 with SAL, a plasmid with our salicylate reporter system. Liquid cultures were made from the conjugation - unfortunately, there was no growth. We streaked new plates with the old transformation in the hopes that something would finally grow!<br />
<div class="panel" style="background:white;margin-top: 20px;"><br />
<h6>Daily Details:</h6><br />
<b>September 9th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the miniprep yields today - all but colony 5 had yields below 90 ug/uL. Colony 5 had a yield of over 500 ug/uL. Our hypothesis as to why the E. coli was growing so slowly was that the E. coli was stressed from having to express the many nah operon proteins. Evidence suuporting our hypothesis includes - colony 5 had a much larger colony, grew in liquid much faster, came out as lacking the nah operon, and yielded a lot more DNA from miniprepping than the colonies that supposedly had the nah operon.<br />
<br><br><br />
<b>Synthetic River Media:</b><br />
Again, the result of the growth assay was failure. This time, it appeared that the only issue was blanking, as the negative controls were reading higher than the inoculated samples. After some discussion with Dr. Archer, a better blanking method was determined, as well as the addition of longer mixing to the protocol.<br />
<br><br><br />
<b>September 10th</b><br />
<br><br><br />
<b>Fluorescence tests: </b><br />
We dephosphorylated SAL2 digested with SpeI and PstI for one hour, then heat killed at 65degC for 10min. We then ligated overnight with mRFP (also digested with SpeI and PstI) at 16degC in the thermocycler.<br />
However, JG700 and p41k didn’t grow, so we moved the plates to Riley Robb in the hopes that a more controlled incubator meant for Shewenella will help them thrive.<br />
<br><br><br />
<b>September 11th</b><br />
<br><br><br />
<b>Fluorescence tests:</b><br />
Swati transformed 2uL of SAL2_mRFP ligation mixture into WM3064. We also made cultures of JG700, WM3064, and p39k, p40k, and p41k in both JG700 and WM3064. We are hoping to run a plate with both strains on it to get an idea of whether we can see mRFP constitutively produced in JG700 (which is naturally red and may have some background signal), and compare that fluorescence to constitutively produced mRFP in WM3064. Once we have this control data we hope to be able to correlate fluorescence in JG700 with promoter strength.<br />
<br><br><br />
<b>September 12th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb and Tina started cultures of colony #3 from original transformation plate and cultures of JG700 + SAL and JG700. JG700 is Shewanella oneidensis with a ΔmtrB genotype. SAL refers to a plasmid with our reporter system that responds to the presence of salicylate by upregulating expression of mtrB.<br />
<br><br><br />
<b>Fluorescence tests:</b><br />
Cultures of JG700 p39k and p41k didn’t grow. We made new cultures with giant swabs of cells from the plates to see if the plates are dead, and subcultured from all the other cultures started yesterday.<br />
There are also two colonies on our SAL2_mRFP transformation plate, so we made 25mL cultures from these to see if we ligated successfully.<br />
<br><br><br />
<b>September 13th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb started liquid cultures of colonies #3 and #4 from the original transformation plate. Caleb also started a conjugation of JG700 + SAL with colony #3 and JG700 + colony #3. The conjugation was at room temperature on a DAP plate for 16.5 hours.<br />
<br><br><br />
<b>Fluorescence:</b><br />
Swati miniprepped the SAL2_mRFP cultures, and Claire set up a PCR to confirm whether SAL2_RFP was ligated successfully. Unfortunately it doesn’t look like the ligations worked, as neither band is ~4.5kb, which should be the length for SAL2 with mRFP. We think that the old digestions we are using may not be good, so we will start from scratch next time.<br />
<br><br><br />
<b>September 14th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures from yesterday and noted there was no growth, as expected, due to the nah operon stressing the cells. Caleb streaked new plates from the conjugation - the LB agar plates had kanamycin and chloramphenicol for the JG700 + SAL and colony #3 conjugation and just chloramphenicol for the JG700 and colony #3 conjugation. The plasmid in the SAL strain confers kanamycin resistance and the plasmid in colony #3 with the nah operon confers chloramphenicol resistance. The strain of E. coli in colony #3 was auxotrophic for DAP, so the E. coli couldn't grow on the newly streaked plates.<br />
<br><br />
<b>From left: Ladder, SAL2_mRFP (1), SAL2_mRFP (2), mut2 pst1, mut2 miniprep:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d4/2012_09_14_sal2RFP_mut2pst1.jpg/800px-2012_09_14_sal2RFP_mut2pst1.jpg"><br />
<br />
<br><br><br />
<b>Synthetic River Media:</b><br />
Cultures of only JG700 and Sal1 containing cells were started today, for another attempt at the growth assay.<br />
<br><br><br />
<b>September 15th</b><br />
<br><br><br />
<b>Running Reactors:</b><br />
Because maximal current production did not increase, Dylan took down reactors with working electrodes poised at 0.35V with respect the the Ag/AgCl reference electrode in order to free up potentiostat channels so that we can begin characterizing our arsenic reporter strains.<br />
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<h3>September 16th-22nd</h3><br />
Conjugating nah into Shewy: Some colonies were observed from last week's restreaking! A massive 15 colony liquid culture was set up, but to no avail. After five long days, we were forced to conclude that conjugation failed. Sadface. Good thing we had extra 3:1 nah to oriT ligation sitting around, which we transformed into both WM3064 and DH5a, the latter of which we hoped would grow faster. 16 colonies from the Sep 4th ligation were also restreaked. And lo! Every single restreak had colonies the next day, which warranted a massive 16 colony colony PCR (try saying that five times fast). Interestingly, the WM3064 plates grew, but the DH5a transformation failed, which we concluded was due to a bad electrocompetent cell stock (we noticed weird white precipitate in the cell mixture before).<br />
<div class="panel" style="background:white;margin-top: 20px;"><br />
<h6>Daily Details:</h6><br />
<b>September 16th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the streaked conjugation plates - there were some colonies, but they were all on top of the initial streak. He and Tina started liquid cultures of all 15 colonies.<br />
<br><br><br />
<b>Synthetic River Media:</b><br />
After finalizing the growth assay protocol, another experiment was set up, this time with only two strains (JG700 and Sal1) in triplicate with sodium lactate concentrations (varying from none to full M4 media concentrations) in synthetic Athabasca river water, plus positive LB controls and blanks. The assay was set to run at room temperature for 20 hours, taking data every 5 minutes with 30 seconds of mixing before each read. Blanking was done by group, for each media mixture.<br />
<br><br><br />
<b>Fluorescence tests:</b><br />
Started liquid cultures of JG700, p25a, and p29a in order to re-conjugate these constitutively produced mRFP plasmids into Shewanella. p25a on a pBBRBB backbone in WM3064 corresponds to p39k in JG700, and p29a corresponds to p41k.<br />
<br><br><br />
<br />
<b>September 17th</b><br />
<br><br><br />
<b>Running Reactors:</b> After autoclaving a reactor setup and getting peristaltic pumps ready for a new continuous flow experiment, Dylan started a liquid culture of an arsenic reporter strain (JG700 + p14k) to be inoculated into reactors.<br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started yesterday - no growth.<br />
<br><br><br />
<b>Synthetic River Media:</b><br />
The growth assay was successful, with constant and low OD600 in the blank wells, and a standard growth curve in the LB wells. The data suggests that lowering the sodium lactate from M4 concentrations is impossible. Therefore, a solution containing 5% volume/volume of 40% sodium lactate weight/volume is what is necessary to maintain an OD600 of approximately 0.1 for an extended period of time. However, the supported OD600 may be higher in the final device at this concentration, due to the constant flow of new sodium lactate.<br />
<br><br><br />
<b>Fluorescence tests:</b><br />
We redigested SAL2 and mRFP with SpeI and PstI-HF to try to clone mRFP into our salicylate reporter, so that we can do fluorescence experiments with the two salicylate parts. Swati also plated WM3064 with JG700 for conjugation of the constitutively produced mRFP.<br />
<br><br><br />
<b>September 18th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started on the 16th - no growth.<br />
Fluorescence tests: We ran mRFP on a gel and extracted a 800bp band which was the appropriate link for mRFP. After enzyme purifying the SAL2 digestion and dephosphorylating, Claire set up an overnight ligation of SAL2 and mRFP.<br />
<br><br><br />
<b>September 19th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started on the 16th - no growth.<br />
Fluorescence tests: Claire transformed the SAL2_mRFP ligation into WM3064, and set up cultures of p39k and p41k from the conjugation plate.<br />
<br><br><br />
<b>September 20th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started on the 16th - no growth.<br />
Fluorescence test: Swati miniprepped cultures of p39k and p41k from the conjugation plates so that we can PCR to confirm if we have successfully conjugated constitutive mRFP into Shewanella.<br />
<br><br><br />
<b>September 21st</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb checked the liquid cultures started on the 16th - no growth. He concluded that the conjugation failed. Caleb and Tina electroporated the 3:1 (nah:oriT backbone) ligation of dephosphorylated and digested nah with the oriT backbone from September 3rd into both WM3064 and a DH5α strain. They then streaked 16 colonies from the September 4th transformation onto fresh plates.<br />
<br><br><br />
<b>September 22nd</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb and Tina noted the appearance of the re-streaked plates - every single streak had numerous colonies. They started liquid cultures and started an overnight colony PCR of all 16 of them. Included with the colony PCRs was a positive control that we knew would have the same amplicon from the nah operon if the colonies were the result of a successful ligation. The electroporated WM3064 plate grew with numerous isolated colonies; the DH5α transformation failed. We hypothesized the DH5α cells were at fault due to their appearance before electroporation - white precipitate was floating in the cell mixture.<br />
<br><br><br />
<b>Fluorescence tests:</b> Using samples from the 20th, Swati did a Phusion PCR to see if we successfully conjugated p39k and p41k into Shewanella. She also miniprepped WM3064 SAL2RFP and did PCR to see if that ligation was successful.<br />
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<h3>September 23rd-30th</h3><br />
Conjugating nah into Shewy: A gel of last week's colony PCRs hinted that one of the colonies had the nah operon in it! Excitement! Liquid cultures were made, and there was just enough DNA after miniprepping to submit for sequencing. We will collectively hold our breaths and wait for positive results.<br><br><br />
This week, we were able to begin characterizing the current response to arsenic of S20 (JG700 + p14k; see strain list). Additionally, we submitted physical DNA for six BioBrick parts to the parts registry.<br />
<div class="panel" style="background:white;margin-top: 20px;"><br />
<h6>Daily Details:</h6><br />
<b>September 23rd</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb and Tina ran a gel of the colony PCRs and positive control PCR. Swati also started cultures of our arsenic-sensitive strain, as well as control strains, for a plate testing our arsenic reporters.<br />
<br><br />
<b>From top-left: ladder, positive control, 8 test colonies. From bottom-left: ladder, 8 test colonies, and Swati's SAL2 RFP:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/2012_9_23_another_colony_PCR.jpg/703px-2012_9_23_another_colony_PCR.jpg"><br />
<br><br />
<b>From left: ladder, empty, p39k PCR, p41k PCR, empty, SAL2_mRFP PCR:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/e/ef/2012_09_23_FloFloFlo.jpg/496px-2012_09_23_FloFloFlo.jpg"><br />
<br />
<br><br><br />
<b>September 24th</b><br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb inoculated 30 mL LB+DAP+Cm with the 24 hour liquid culture of of colony N.<br />
<br><br><br />
<b>September 25th</b><br />
<br><br><br />
<b>Fluorescence tests:</b> Swati set up a conjugation of SAL2_mRFP into JG700. Also, sequencing came back good so once we have this part in Shewanella we can start testing our salicylate sensor. Jim and Swati also set up a plate to test our arsenic sensing parts at different concentrations of arsenic – they ran a plate with a blank LB column, five control columns (JG700, MR-1, p39k, p40k, and p41k), and three columns of each of our arsenic-sensitive strains. To each row they added a different concentration of arsenic, going from 0uM to 5uM arsenic. The final OD of 100uL in each well was 0.8, and the plate was left overnight in the plate reader.<br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Caleb miniprepped the 36 hour 30 mL culture of colony N. The size of the pellet after the first spin step was similar to the size of a ~5mL normal culture, consequently, the yield was only 96.7 ng/uL.<br />
<br><br><br />
<b>September 27th</b><br />
<br><br><br />
<b>Running Reactors:</b> For the first time, we added arsenic to our reactors. Specifically, once Dylan saw that the two reactors inoculated with an arsenic reporter strain (JG700+p14k) had been producing steady current for several retention times, he dosed the media vessel to a final concentration of 10 μM sodium arsenite.<br />
<br><br><br />
<b>Conjugating nah into Shewy:</b><br />
Fortunately, there was enough DNA for Caleb to submit the miniprepped DNA from colony N for Sanger sequencing today. Tina started 3 x (1mL cultures of colony N).<br />
<br><br><br />
<b>Fluorescence tests:</b> Claire and Swati set up another control plate with constitutively produced mRFP in both WM3064 and JG700. Since using 100uL starting at and OD of 0.8 seemed to work well for the arsenic plate, we set up the control plate with the same parameters and left it in the plate reader overnight.<br />
<br><br><br />
<b>September 28th</b><br />
<br><br><br />
<b>Running Reactors:</b> Since several retention times passed without any current response to 10 μM sodium arsenite, Dylan increased the concentration in the media vessel, hitting our reactors with 100 μM arsenite.<br />
<br><br><br />
<b>Fluorescence tests:</b> We noticed that the arsenic data did not seem to have any signal above background, and realized that it may have been because the plate reader wasn’t set to read a clear-bottomed plate. We concluded that, since from the test done on the 27th we could see distinct difference between fluorescence levels of constitutively produced mRFP, our experimental procedure is good and the plate reader was just on the wrong setting. Additionally, since the bioreactors are not getting upregulation of arsenic up to 10uM, we decided next time to test higher concentrations of arsenic (0uM to 500uM)<br />
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<h2 class="centered">Wet Lab - August</h2><br />
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<h3>August 1st - 4th</h3><br />
It was confirmed that RFP was successfully inserted downstream of mtrB in our arsenic reporter parts in Shewanella. Site-directed mutagenesis of the nah operon failed. We are still trying to insert the nah operon into the mobility backbone (OriT) to prepare for conjugation into Shewanella later on. <a href="#" class="technical-desc" for="#technical-desc1" style="display:block;margin-top:20px;">Daily Details</a><br />
<div class="hide-me panel" style="background:white;margin-top: 20px;" id="technical-desc1"><br />
<h6>Daily Details:</h6><br />
<br><b> August 1st, Wednesday </b></br><br> In the morning, Dylan noted that 5 of the 7 transformations from Tuesday produced plates with colonies. Those that worked were three Anderson series constitutive promoters (with downstream mRFP) in pBBRBB and the versions of our two arsenic reporters with mRFP downstream of mtrB. In the evening, Swati and Dylan prepared overnight cultures from all transformants and JG700, both to set up conjugations to get plasmid into Shewanella and to screen isolated plasmids for successful ligation. We will use these constructs to characterize the activity of the arsenic-sensitive promoter in Shewanella with respect to known constitutive promoter strengths. The two transformations that failed were versions of the salicylate reporter. <br />
After staring at plates, Dylan ran the nah+p29, oriT+p17c, p31c, and nah operon (from p20) digestions on a gel, extracted the fragments of interest, and then dephosphorylated the backbone. The isolated digestion products were used later used by Steven, who set up two overnight ligations – one to put the nah operon, with a constitutive promoter, on a pSB3C5 backbone with an added oriT sequence, and one to put the nah operon into the MCS of pSB1C3 for subsequent site directed mutagenesis. <br />
While Dylan ran his gel, Caleb miniprepped from the JG700+SAL and S31 cultures. After quantification, SAL plasmid isolated from JG700 was submitted for sequencing in order to confirm successful conjugation. <br />
Meanwhile, Mark decided that he enjoyed making DH5a electrocompetent stocks on Tuesday so much that he had to make more WM3064 electrocompetent stocks today. Starting from subcultures that Dylan had prepared, he prepared the stocks, and was assisted by Danielle. <br />
<br><br />
<b>From left: ladder, oriT+p17c digest (E+X), nah + p29 digest (E+S), p31c digest (E+S), p20 PCR digest (E+S):</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/5/5d/2012_08_01_nahsDigest.jpg/800px-2012_08_01_nahsDigest.jpg"><br />
<br />
</br><br />
<br><b> August 2nd, Thursday </b></br><br> Caleb and Dylan created conjugation plates of p14RFP, p16RFP, and p25-29BB as well as miniprepped from the same plasmids in WM3064 (see: strain list). Dylan, Mark, and Tina then desalted and transformed SAL2 and SALRFP into WM3064 as well as p31c_nah and oriT_nah_p17c into DH5a. Tina and Dylan then quantified the minipreps from earlier in the day. Dylan also digested pBBRBB and p33k with EcoRI &amp; PstI to put lac inducible mtrB into the pBBRBB backbone.<br />
</br> <br> </br><br />
<br />
<br><b> August 3rd, Friday </b></br><br> After running a digestion of p33k, the lac inducible mtrB part from the parts registry, we discovered that the part does not include mtrB! We should have seen a ~3.6kb band on our gel after digesting with EcoRI and PstI, but instead isolated a band ~1.5kb. After checking the sequencing on the parts registry we discovered this was because the part didn't include mtrB, so we won't be able to use it as a positive control for inducible mtrB. <br />
Sequencing of p37k-p41k from WM3064 came back good, meaning we can put these control parts, with RFP downstream of our various reporters, into Shewanella via conjugation. Hence, conjugation was carried out. <br />
<br><br />
<b>Digests of p11 and p33 (E+P):</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/6/69/2012_08_03_p11p33dig.jpg/800px-2012_08_03_p11p33dig.jpg"><br />
<br />
</br> <br> </br><br />
<br />
<br><b> August 4th, Saturday </b></br><br> This week, it was confirmed that RFP was successfully inserted downstream of mtrB in our arsenic reporter in Shewenella. <br />
Site-directed mutagenesis of the nah operon failed. We continued trying to insert the nah operon into the mobile backbone (OriT) to prepare for conjugation into Shewanella. <br />
</br> <br> </br><br />
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<h3>August 5th - 11th</h3><br />
This week, we continued our site directed mutagenesis of nah_p31c and the addition of RFP to our SAL reporters. Sequencing of the arsenic reporter has been unsuccessful so far. We also decided that we need more quantitative data to characterize our parts.<br />
<a href="#" class="technical-desc" for="#technical-desc7" style="display:block;margin-top:20px;">Daily Details</a><br />
<div class="hide-me panel" style="background:white;margin-top: 20px;" id="technical-desc7"><br />
<h6>Daily Details:</h6><br />
<br><b>August 5th, Sunday </b></br><br> Swati yet again was saddled with a massive number of <a href="https://static.igem.org/mediawiki/2012/3/35/Ezna_Miniprep.pdf">minipreps </a>, since her magic fingers are able to cast yield-increasing spells on minipreps. She miniprepped: SAL2 from WM3064, p37-41k from JG700 (one arsenic reporter with cut sites flanking the RBS, another arsenic reporter without the cut sites, and three different Anderson series promoters with mRFP1 downstream on a pBBRBB backbone), oriT p29nah_p17c from DH5a, and nah_p31c from DH5a. And indeed her yields were impressive, massive, gargantuan! Good job Sorceress Swati. <br />
We will <a href="https://static.igem.org/mediawiki/2012/0/03/Sequencing_Preparation.pdf "> sequence </a> SAL2 and oriT p29nah_p17c to see if we should continue to conjugation, and sequence nah_p31c to see if we can start <a href=" https://static.igem.org/mediawiki/2012/a/a5/Site_Directed_Mutagenesis.pdf "> site-directed mutagenesis </a> to get a biobrick-compatible nah operon on the biobrick backbone. We will sequence or PCR to confirm p37k-p41k's conjugation into Shewanella. <br />
</br> <br> </br><br />
<br />
<br />
<br><b> August 6th, Monday </b></br><br> Caleb and Dylan amplified the two different arsenic promoters (p37k and p38k) and ran them on a gel alongside p14 and p16 (the arsenic reporters without mRFP1 downstream) to confirm successful insertion of RFP downstream of mtrB in our arsenic reporter parts in Shewenella. Band lengths appeared in expected places. Dylan is worried that we will not be able to use PCR to definitely confirm that p39-41k worked, so we submitted these for <a href="https://static.igem.org/mediawiki/2012/0/03/Sequencing_Preparation.pdf "> sequencing </a>. Unfortunately, sequencing failed, perhaps because random gunk from Shewanella added noise to the process. <br />
Tina did a PCR cleanup of the nah operon PCR. Dylan also ran a gel of nah operon PCRs to make sure that there was no mispriming. The gel looked good, so he submitted the PCRs for sequencing. <br />
<br><br />
<b>Successful insertion of RFP downstream of mtrB in arsenic reporter strains in Shewanella:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/2012_08_06_p37p38verify.jpg"><br />
<br />
</br> <br> </br><br />
<br />
<br />
<br><b> August 7th, Tuesday </b></br><br> JG1531 overnight culture didn't grow. We suspect that the plate Dylan picked from is dead. We'll have to go back to the glycerol stocks if we want to play with mtrE. Dylan set up a continuous flow M4 reactor in morning. Caleb started a liquid culture of MR-1 with which to inoculate the reactor. Checked sequencing results of nah stuff. oriT thing was bad. nah_p31c was good. <br />
Caleb and Dylan proceeded with <a href=" https://static.igem.org/mediawiki/2012/a/a5/Site_Directed_Mutagenesis.pdf "> site-directed mutagenesis </a> of nah_p31c, attempting to get rid of the first PstI cutsite in the nah operon. After digestion with DpnI, we purified using the E.Z.N.A. MicroElute kit and quantified. DNA was split into three directions: First, Danielle and Dylan set up another mutagenic PCR using the digestion product as template to get rid of the second internal cut site (we expect lower mutation efficiency because template DNA is not methylated). Second, we transformed DH5a with the mutated plasmid. Third, Danielle and Chie <a href="https://static.igem.org/mediawiki/2012/a/af/Double_Digest.pdf "> digested </a> both the purified – and hopefully mutated – plasmid and un-mutated plasmid with PstI. Dylan ran these digestions on a gel, along with supercoiled plasmid as a control. Unfortunately, the (hopefully) mutated plasmid never showed up on the gel. <br />
In the evening, we didn't observe growth in the MR-1 culture, so Dylan set up another culture just in case Shewanella was dead and not lazy. <br />
<br><br />
<b>Looking for mutagenesis – from left – ladder, unmutated nah, mutated nah, supercoil control:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d9/2012_08_07_mutNAHcheck.jpg/800px-2012_08_07_mutNAHcheck.jpg"><br />
<br />
</br> <br> </br><br />
<br />
<br />
<br><b> August 8th, Wednesday </b></br><br> Caleb and Dylan performed another <a href="https://static.igem.org/mediawiki/2012/a/af/Double_Digest.pdf "> digest </a> to check if the second <a href="https://static.igem.org/mediawiki/2012/a/a5/Site_Directed_Mutagenesis.pdf "> site-directed mutagenesis </a> (that was supposed to get rid of the second PstI internal cut site within the nah operon) worked. Unfortunately, there wasn't enough DNA to see anything when visualized on a gel. They decided to proceed with electroporation into DH5a just in case - however, to use Caleb's terminology, cells exploded - there were probably too many salts in the solution, which causing arcing and a PBBHTTTZZZ! of cells all over the cuvette. <br />
Dylan re-digested p29 nah and oriT p17c to redo a ligation that would allow for conjugation into Shewanella later on. Dylan also re-digested p27 (Anderson series promoter with RFP downstream) and SAL to redo a ligation to create a plasmid with SAL-RFP. <br />
Dylan also submitted several samples for <a href="https://static.igem.org/mediawiki/2012/0/03/Sequencing_Preparation.pdf "> sequencing </a> to make sure that three Anderson promoters with RFP downstream in JG700 and SAL2 (the salicylate reporter without the BAMHI cut site) in WN3064 and JG700 were in the correct sequence. <br />
<br><br />
<b>Seeing if mutagenesis worked; nothing there:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/6/6a/2012_08_08_nothingthere.jpg/800px-2012_08_08_nothingthere.jpg"><br />
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</br> <br> </br><br />
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<br><b> August 9th, Thursday </b></br><br> Today Dylan chilled with his mom. Well, he tried to. :) Despite the eternal bonds that bind mother and son, plus the 3 billion miles she flew to see him, he still couldn't resist coming in to lab to open packages! And then got sucked into a long discussion of what had to be done for the rest of the day - scientific endeavors taking him away yet again from filial duties. Swati and Claire bonded over dry ice, and Caleb regaled us with tales of dry ice bombs gone awry. <br />
Swati and Caleb miniprepped the first mutagenesis of nah_p31c that had been transformed into DH5a yesterday. They then digested it with PstI-HF to see if the mutation was successful. Unfortunately the mutagenesis failed! We will start over from nah_p31c and lower the annealing temperature at 60degC. The Stratagene kit, which uses PFU ultra, asks for 60degC, but because we are using our own boot-leg protocol with Phusion we did the first try at 65degC, as Phusion usually calls for a higher annealing temperature than the theoretically calculated value. For this second try we will stick to the 60degC suggested by Stratagene and see if we get better results. Also, called NEB to find out if after PCR with Phusion, DpnI will still have activity in the following digestion step in Buffer 4, or if we need to clean up the PCR before digesting with DpnI - they said PCR clean up isn't needed. <br />
We also ran digestions for making the nah operon on a backbone with a mobility gene and the salicylate reporter (w/ BamHI cutsite) with RFP downstream. We cut our mobile backbone, OriT in p17c (pSB3C5), with EcoRI and XbaI, while cutting the nah operon (p29nah) with EcoRI and SpeI. The nah operon with mobility gene must be constructed so that we may conjugate into Shewy and start testing our salicylate reporter. The salicylate reporter and p27a (from the Anderson series), with RFP, were cut with SpeI and PstI. The salicylate/RFP part will be used for troubleshooting the salicylate reporter. Digestion products were run on a gel, extracted, and quantified. Swati then dephosphorylated backbones and ligated both parts. <br />
Sequencing for p39-41k didn't look good, and neither did the salicylate reporter w/BamHI cutsite miniprepped from JG700. The sequencing for salicylate reporter w/ BamHI in WM3064, however, looked good, suggesting that conjugation may not have been as efficient as we had hoped. After a pow-wow we decided not to sequence more colonies, as we are hoping some may be good, and more importantly that if we use qPCR to get quantitative characterization data, we won't need to use the RFP parts. The plates will stay in the fridge as a back-up plan. <br />
In other news: Claire cried because it was difficult to update the notebook with a week's worth of work. Mark's dedication to the notebook is laudable and impressive. Good job team for doing so much! My head can't even comprehend the magnitude of your endeavors. <br />
<br><br />
<b>Unsuccessful mutagenesis (test with Pst1 cutsite):</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/8b/2012_08_09_pstI_cut_site_test.jpg/800px-2012_08_09_pstI_cut_site_test.jpg"><br />
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<b>Digestion products from left – ladder, p27, p29nah, oriTp17c, SAL:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/1/1e/2012_08_09_SALRFP_nah17_dig_copy.jpg/534px-2012_08_09_SALRFP_nah17_dig_copy.jpg"><br />
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</br> <br> </br><br />
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<br><b> August 10th, Friday </b></br><br> Caleb miniprepped a plasmid with mtrE from JG1531, and just for kicks, miniprepped from the "exploded cells" from August 8th. Suprisingly, he ended up getting decent yields for both, showing that the electroporation worked despite arcing. Caleb then digested the nah operon of the miniprep with PSTI and NotI to check if the mutagenesis was a success. It was run on a gel alongside a PCR of the Anderson series promoters with RFP downstream and SAL2 from Shewanella (to check if SAL2 and the promoters were sucessfully conjugated after sequencing on Monday failed). Unfortunately, bands did not appear where we expected them to. <br />
Steven and Spencer performed a PCR to get mtrE out of the Gralnick (JG700) plasmid.<br />
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<b>From left lane: ladder, p39 PCR, p40 PCR, p41 PCR, nah-p31c (P+N digestion), SAL2 PCR:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/df/2012_08_10_longer_exposure.JPG/800px-2012_08_10_longer_exposure.JPG"><br />
</br> <br> </br><br />
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<br><b> August 11th, Saturday </b></br><br> Spencer and Steven checked the nah operon PstI and NotI digest (because yesterday's gel ran weirdly) and their mtrE PCR from the previous day on a gel. Unfortunately, bands did not appear where we expected them to.<br />
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<b>mtrE PCR product (left) and checking mutagenesis again (right):</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/7/7e/2012_08_11_mtrE%2C_digest.JPG/800px-2012_08_11_mtrE%2C_digest.JPG"><br />
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<h3>August 12th - 18th</h3><br />
This week, we: <br />
1)Performed the neccessary work for getting our reporters into the pSB1C3 backbone required for submission. This included the neccessary digestions of our reporters out and ligations into the submission plasmid. Confirmation of successful transformation and ligation will be performed in the following weeks. <br />
2)Confirmed successful transformation of Shewanella with our arsenic and salicylate reporters via colony PCR. <br />
3)Performed the neccessary digestions and ligations to have mRFP downstream of our reporter system so as to do an additional fluorescent testing of the reporters to serve a second form of confirmation of increased transcription in the presence of our toxins. <br />
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<a href="#" class="technical-desc" for="#technical-desc2" style="display:block;margin-top:20px;">Daily Details</a><br />
<div class="hide-me panel" style="background:white;margin-top: 20px;" id="technical-desc2"><br />
<h6>Daily Details:</h6><br />
<br><b> August 12th, Sunday </b></br><br> Weekend overview: It looks like the mtrE primers are actually working to amplify the part out of JG1531. We'll be putting mtrE in p31c, both so that we may submit the novel part to the registry, and to begin site-directed mutagenesis. <br />
We'll use mtrE mutagenesis as something of a control for nah operon mutagenesis (to see if the large size of the plasmid and nah operon is the problem). <br />
It also looks like nah operon mutagenesis hasn't worked. We'll put this on hold, and continue with ligation of p29nah into oriT_p17c once desalting paper arrives. <br />
Also, we'll have to redo things to confirm p39-41, SAL2 in JG700. What was done over the weekend looks weird. <br />
</br> <br> </br><br />
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<br><b> August 13th, Monday </b></br><br> Dylan ran four simultaneous Phusion PCRs with an annealing temperature of 67degC and an extension time of 45sec to amplify mtrE. The gel looked good, with no mispriming and one band ~2.2kb. Claire did PCR clean up of the product, which will eventually be cut and ligated into p31c (see: strain list). <br />
Because the results from the weekend's screenings were confusing, we also ran PCRs of p39-41k (miniprepped from JG700 strains) to confirm whether conjugation into Shewanella was successful. These plasmids contain mRFP under the control of constitutive promoters of varying strengths. After confirmation, we will use these parts to characterize our inducible promoters in Shewanella. <br />
Dylan also started a liquid culture of JG700 + SAL to be inoculated into a continuous flow reactor with M4 media. With this setup, we will begin characterizing our reporter strains, initially in response to salicylate. <br />
Dylan performed a Phusion PCR to amplify mtrE, and ran a gel to check for product. There was a single band so no mispriming was occurring. <br />
Claire performed a double digest of the p14k and p16k with XbaI and PstI HF. This is for the construction of our final biobricks to be submitted to the registry. <br />
<br><br />
<b>PCR amplification of mtrE:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c0/2012_08_13_mtrE%2C_PCR.JPG/536px-2012_08_13_mtrE%2C_PCR.JPG"><br />
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<b>p39 and p41 verification:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/f/f1/2012_08_13_p3941confirm_overnight.jpg/520px-2012_08_13_p3941confirm_overnight.jpg"><br />
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<br><b> August 14th, Tuesday </b></br><br> Steven performed a double digest of mtrE (EcorI &amp; SpeI), Sal (SpeI &amp; PstI), Sal2 (SpeI &amp; PstI), and p26a (SpeI &amp; PstI). <br />
Steven performed a ligation of p29nah + oriTp17c, and also a ligation of p27 digest + Sal. Dylan transformed colonies with the ligation. <br />
</br> <br> </br><br />
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<br><b> August 15th, Wednesday </b></br><br> Dylan ran a gel and Claire performed gel extraction of mRFP to put downstream of Sal reporters. They also checked to see if site-directed mutagenesis was successful for the nah-p31c. Found the digest of mRFP and p26 looked good, but the mutagenesis looked strange. <br />
Dylan performed a taq PCR to confirm the presence of Sal2 in Shewanella. However no band was present in the gel ran afterwards, so perhaps colony PCR is the next best step. <br />
Dylan also ran a gel of the p14k, p16k p31 digests (all cut with XbaI and PstI) for the set-up for forming the biobricks. <br />
Steven performed a colony Phusion PCR of four colonies containing the p4k plasmid to again test for the presence of mtrE. Spencer then did PCR clean up <br />
Spencer performed a double digest of p4kdlg and p27a cutting with EcoRI &amp; SpeI followed by running a gel and extracting the digests. <br />
Spencer picked colonies from the previous ligations of the Sal, Sal2, p31c biobrick plasmids for miniprepping on Thursday. <br />
This morning Dylan noted that we may be getting a detectable basal level of current - around 6mA - in a continuous flow setup. This would make it possible to bypass the addition of mtrE to the salicylate sensing part, and still be able to distinguish Shewanella not detecting salicylate from dead Shewanella. He then added salicylate to the reactor innoculated with our salicylate reporting strain, bringing the concentration to 10uM. Later in the day, he noted that the current had risen to around 9mA, which is promising: our strain may be working! Claire set up four hour digestions with XbaI and PstI to put our arsenic parts, p14 and p16, into the iGEM backbone for submission. <br />
Dylan also transformed a salicylate reporter with mRFP downstream into DH5alpha and WM3064, as well as the nah operon with the p29 promoter in a p17c backbone with an oriT. The second of these could be conjugated into Shewanella if transformed successfully. The first, the salicylate reporter with mRFP downstream, is a fluorescent version of the salicylate reporter. The fluorescent versions of our reporters is an alternative to qPCR to measure the relative expression level of mtrB: if we know what mRFP under the control of a constitutive promotor in Shewanella looks like, we can add arsenic or naphthalene/salicylate to our fluorescent reporter parts until reaching the same level of expression. Then, we can correlate this concentration of arsenic or salicylate to a certain strength of induced expression. <br />
In case the fluorescent SAL reporter was not successfully ligated, we are preparing more mRFP, SAL, and SAL2: - In the morning Claire also cleaned up the digestions of SAL, SAL2 and mtrE PCR. However, due to a silly mistake on her part involving wash buffer and absolute ethanol (absolutely missing, to be precise) we will redo the digestions in order to get more DNA for putting these reporters into the iGEM backbone, and to put mRFP downstream. She then set up four hour digestions with XbaI and PstI to put our arsenic parts, p14 and p16, into the iGEM backbone for submission. - We digested mRFP with SpeI and PstI to make the part with mRFP downstream of mtrB in the salicylate reporters. We have already successfully put mRFP downstream of mtrB in our arsenic reporters, but it did not seem to work in the arsenic reporter. The gel for mRFP looked as expected, with the band for the insert slightly shorter than 900bp. However, on the same gel the the mutagenesis trial of the nah operon, digested with PstI and NotI, showed only one band. If mutagenesis had not worked, we would expect more than two bands, and if it had worked we would still expect two bands. Therefore we are unsure how to interpret this result, which we have seen twice now, but are going to start over with site-directed mutagenesis of the nah operon. We will also visualize the unmutated nah operon, digested with PstI and NotI, on a gel to see if that looks like we expect it to. <br />
Finally, it should be noted with jubilance that Claire was reunited with her umbrella today! The joy in the lab at this event was palpable and will be remembered for years to come.<br />
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<b>mRFP digest and additional (failed) attempt at confirming mutagenesis:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d9/2012_08_15_mRFPdigest_mutnahPNDigest.JPG/583px-2012_08_15_mRFPdigest_mutnahPNDigest.JPG"><br />
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<b>From left – ladder, failed SAL2, p14, p16, p31digestions:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c5/2012_08_15_sal2fail_p14_16_31_dig.jpg/689px-2012_08_15_sal2fail_p14_16_31_dig.jpg"><br />
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</br> <br> </br><br />
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<br><b> August 16th, Thursday </b></br><br> Dylan performed a miniprep of the Sal, Sal2, and p31c reporters for us to do sequencing for confirmation of successful ligations. These will hopefully be the biobricks we send in. <br />
In the afternoon Dylan performed a dephosphorylation of the p31c (cut with XbaI, PstI) followed by a gel of the mtrE cut with EcoRI &amp; SpeI as well as mRFP cut with SpeI and PstI. <br />
A 4 hour digestion was also performed by Claire to get Sal parts into pSB1C3 for the SAL, SAL2, and p31 parts. Steven then ran a gel of the digest products. <br />
Steven also performed a colony Taq PCR of the Sal2 reporter to confirm its presence in Shewanella. <br />
Today, we continued work to get our engineered reporters into pSB1C3 for submission to the parts registry. In the morning, Dylan miniprepped both versions of our salicylate reporters, along with more pSB1C3 from overnight cultures of the corresponding DH5a strains. Following quantification, he set up digestions of each miniprep with XbaI and PstI. (We decided to cut with XbaI because we discovered an extra base pair between the NotI and XbaI cutsites in the normal BioBrick prefix, an artifact of a previous team's work). Caleb also dephosphorylated previously isolated pSB1C3 backbone to prevent self-ligation, while Steven gel extracted the salicylate reporter inserts and more pSB1C3 backbone (to be quantified Friday morning). <br />
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<b>mtrE and mRFP digests:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/86/2012_08_16_mtrEdig_mRFPdig.jpg/557px-2012_08_16_mtrEdig_mRFPdig.jpg"><br />
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<b>Digestions of SAL, SAL2, and p31:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/7/7f/2012_08_16_SALSAL2p31dig.jpg/553px-2012_08_16_SALSAL2p31dig.jpg"><br />
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<br><b> August 17th, Friday </b></br><br> Claire performed a ligation for the construction of the Sal_RFP, and Sal2_RFP plasmids. Transformation was then performed immediately after.<br />
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<h3>August 19th - 25th</h3><br />
Conjugating nah into Shewy: <br />
Colony PCR was done to 1) make sure the nah operon was in the cells 2) troubleshoot the colony PCR protocol After imaging, we discovered that the colonies did contain the nah operon, and that our colony PCR protocol was good.<br />
<a href="#" class="technical-desc" for="#technical-desc3" style="display:block;margin-top:20px;">Daily Details</a><br />
<div class="hide-me panel" style="background:white;margin-top: 20px;" id="technical-desc3"><br />
<h6>Daily Details:</h6><br />
<br><b> August 19th, Sunday </b></br><br> In the morning, Dylan came to lab to look at the transformant plates from Saturday. There were colonies on all but one of the plates: It looks like either the ligation to put mtrE into p31c or the subsequent electroporation failed. It is of note that the competent DH5α cells that Dylan used for the mtrE electroporation were from an older stock – one which we'd previously determined to confer lower transformation efficiency. Because we used up all of the newer stock – with markedly better efficiency – we will make more electrocompetent DH5α in the coming week.<br />
Because all other transformations seemed to work, we grew up overnight cultures from single colonies on each plate. We will miniprep from these cultures and screen the minipreps for the correct insert. If confirmed, we will have successfully have put both versions of our arsenic reporters, as well as both versions of our salicylate reporters inside p31c (i.e., pSB1C3), as is required for registry submission. We will also have successfully put mRFP downstream of mtrB on SAL2, which will be used for characterization of the salicylate sensitive promoter in S. oneidensis.<br />
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<b>Gel showing failure of nahoriT p17c:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/6/6d/2012_08_19_p29nahoriTp17cfail.jpg/584px-2012_08_19_p29nahoriTp17cfail.jpg"><br />
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<br><b> August 22th, Wednesday </b></br><br> Conjugating nah into Shewy: Caleb, Tina, and a guest Louis miniprepped 5 plasmids containing nah operons with constituitive promoters from a WM3064 plate and miniprepped one plasmid that we previously confirmed as having the nah operon (without a promoter) to use as a positive control in the future. <br />
Claire performed miniprep of grown cultures of the Sal_RFP and Sal2_RFP to submit for sequencing. <br />
</br> <br> </br><br />
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<br><b> August 24th, Friday </b></br><br>Jim performed the digestion of the nah operon+ p29a and the p31c backbone for ultimate ligation in the creation of the last biobrick. Ligation had to be redone, as sequencing results of last week's ligations looked great except for the nah operon biobrick. <br />
<br />
Conjugating nah into Shewy: <br />
We wanted to accomplish two things: <br />
• Amplify the nah operon with a promoter using primers with appended cut sites. <br />
• Optimize colony PCR for future Cornell iGEMers (past attempts have failed.) <br />
To accomplish both of these objectives, we did Phusion PCRs using plasmids containing nah operons with constituitive promoters as a template and a Phusion colony PCR using the same colonies we miniprepped the plasmid from. Because we expect the miniprep PCRs to work, doing the colony PCRs in parallel will allow us to determine whether our colony PCR technique is working or not. For our colony PCR purposes, we want to be able to screen for colonies while being certain we can go back and miniprep the same PCRed colony. Our colony PCR method is as follows: <br />
NOTE: We later determined there was a cataclysmic flaw in the following method, can you figure it out? (wink wink nudge nudge...) <br />
• Label colonies of interest and correspondingly label microfuge tubes <br />
• Add 50 uL ddH2O to each microfuge tube <br />
• Using pipette tip, dip into colonies of interest then dip into labelled microfuge tubes and swirl around to release cells <br />
• Use the the ddH2O+cell mixture as the DNA template for a PCR - instead of adding plain ddH2O to the PCR to bring it up to volume, add the ddH2O+cell mixture <br />
• Confirm your PCR had desired results with a DNA gel, then use the ddH2O+cell mixture from the corresponding microfuge tube the PCR was done on to inoculate a culture or plate. <br />
</br> <br> </br><br />
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<br><b> August 25th, Saturday </b></br><br> Jim performed an overnight ligation of the nah operon pSB1C3 for transformation on Sunday. <br />
Conjugating nah into Shewy: <br />
Caleb, Tina, and a guest Louis ran a gel of the miniprep and colony parallel Phusion PCRs. All of the miniprep PCRs worked, but only a couple colony PCRs worked. We believe this is due to not having added enough cells to the colony PCR tube because some of the colonies were very tiny. <br />
<br><br />
<b>Top: Positive control from colony, colony test 1, colony test 2, colony test 3, colony test 4, colony test 5, miniprep 5, Benchtop 1kb; Bottom: Benchtop 1kb, miniprep 1, miniprep 2, miniprep 3, miniprep 4, positive control miniprep:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c9/2012_8_25_Checking_for_nah_product_from_colony_vs_miniprep_PCRs.jpg/489px-2012_8_25_Checking_for_nah_product_from_colony_vs_miniprep_PCRs.jpg"><br />
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<h3>August 26th - 31st</h3><br />
Operation “get nah into Shewy continued this week.” We were not able to confirm through colony PCR that we have nah in WM3064. Team fluorescence was able to conjugate the SAL_mRFP constructs into Shewy. We continued site-directed mutagenesis of the nah operon but it has not been working like we expected. <br />
<a href="#" class="technical-desc" for="#technical-desc4" style="display:block;margin-top:20px;">Daily Details</a><br />
<div class="hide-me panel" style="background:white;margin-top: 20px;" id="technical-desc4"><br />
<h6>Daily Details:</h6><br />
<br><b> August 26th, Sunday </b></br><br> Conjugating nah into Shewy: <br />
Caleb miniprepped two plasmids - one contained the nah operon preceded with a constitutive promoter and the other is to become a backbone containing the origin of transfer required for conjugation and a gene conveying chloramphenicol resistance. The purified plasmids were digested to extract the nah operon and to cut open the backbone - these digests were then run on a gel. Tina gel extracted the backbone then accidentally threw away the rest of the gel that contained the nah operon fragments. <br />
<br> Site-directed mutagenesis: Swati ran a mutagenic PCR of nah_p31c, then digested with DpnI to remove template DNA and column purified the resultant DNA. </br><br />
</br> <br> </br><br />
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<br><b> August 27th, Monday </b></br><br> Conjugating nah into Shewy: <br />
Caleb gel extracted the nah operon containing fragments from yesterday - despite having been in the garbage overnight, the gel extraction worked! Caleb and Tina began a 24 hour 16 degrees Celsius ligation using molar rations of nah to backbone of 3:1 and 1:1. <br />
Fluorescence tests: Claire started a JG700 + SAL2 culture and tried to do a PCR of SAL2_mRFP with sequencing primers to see if the band produced would correspond to the length of SAL2 plus the length of mRFP. Unfortunately, due to a mishap involving p10s vs. p2s and not checking what volume her pipette was set to, she ended up with a 70uL total volume and PCR failed. <br />
<br> Site-directed mutagenesis: Swati transformed the (hopefully) mutated DNA into DH5a. </br><br />
</br> <br> </br><br />
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<br><b> August 28th, Tuesday </b></br><br> Conjugating nah into Shewy: <br />
Caleb and Tina de-salted the 3:1 and 1:1 ligations, then transformed them into a DAP-requiring conjugation strain WM3064 via electroporation. The electroporated cells were plated on DAP + Cm plates. <br />
Fluorescence tests: JG700 once again takes a long time to grow, so it wasn’t until the evening that Claire miniprepped SAL2 from JG700. Tomorrow we will run another PCR and see if we have SAL2_mRFP, and SAL2 in JG700. <br />
<br> Site-directed mutagenesis: Swati's transformation did not work. After checking the PCR product and a PstI digest thereof on a gel, she discovered that the PCR was not working with high enough efficiency to produce a band on a gel. Upon consulting with Didi, our trusty post-doctoral adviser, she then restarted the mutagenic process with a PCR; this time, with a lower primer concentration and lower denaturation and annealing times. </br><br />
</br> <br> </br><br />
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<br><b> August 29th, Wednesday </b></br><br> Conjugating nah into Shewy: <br />
Caleb checked the transformation plates after ~14 hours in a 37 degrees Celsius incubator and noticed colonies, but the colonies were barely visible indicating very slow growth of the cells. <br />
Fluorescence tests: Set up a Taq PCR of SAL_mRFP, SAL2_mRFP and SAL2, with an extension time of 4.5 minutes and annealing temperature of 68degC. SAL2_mRFP has a band corresponding only to the length of SAL2, without mRFP, so we need to redo this ligation. However, the band from SAL_mRFP was the correct length (a little more than 4kb), so this ligation was successful and we can conjugate into Shewenella. Finally, the JG700 miniprep is the correct length for SAL2 (~3.8kb), so SAL2 has been successfully conjugated into Shewenella! <br />
<br> Site-directed mutagenesis: Swati digested her second mutagenic PCR with DpnI, column purified, and transformed into DH5a. </br><br />
</br> <br> </br><br />
<br />
<br><b> August 30th, Thursday </b></br><br> Conjugating nah into Shewy: <br />
Tina checked the growth of yesterday's colonies and tried doing a colony PCR of each colony and a PCR of a positive control (same product, same primes, but in a different purified plasmid). The primers were designed to sit on the backbone and face into the oriT region and the site where we wanted to insert the nah operon. A PCR amplicon indicating successful ligation of the nah operon into the backbone would be approximately 10-11kb, while a failed ligation would yield a ~700 bp amplicon. Caleb later ran a DNA gel of the colony PCRs and nothing but smears showed up in every lane of the gel except the positive control which didn't even have a smear (Caleb was so disappointed that he didn't post the picture). Since the positive control didn't show up, we concluded the PCR failed. Caleb started overnight cultures of the remaining cells+ddH2O used for the colony PCR. <br />
<br> Site-directed mutagenesis: Alas, Swati's second attempt at mutagenesis was yet again fruitless. Concerned that the template may have been causing the problem, she performed a NotI digest and ran it on a gel alongside the latest mutagenic PCR. The template was fine; the PCR was, again, invisible. Swati grew extremely puzzled as to how her purified PCR product could have significant concentration upon quantification but yield no visible bands on a gel. She decided to try a third time, this time with longer denaturation and annealing times, but still the same concentration of primers. <br />
<br><br />
<b>From left lane: Ladder, SAL2_mRFP (1), SAL2_mRFP (2), SAL2:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/f/fc/2012_08_30_gel.jpg/800px-2012_08_30_gel.jpg"><br />
<br />
</br><br />
</br> <br> </br><br />
<br />
<br><b> August 31st, Friday </b></br><br> Conjugating nah into Shewy: Caleb noticed the overnight cultures didn't grow and concluded the cells must have died due to the cells being stored in an extremely hypotonic solution (ddH2O). He re-did colony PCRs of the colonies, a colony with a positive control, and a positive control of purified plasmid, but modified the colony PCR protocol. Instead of using the cells + ddH2O as template DNA, Caleb dipped the pipette tip into the colony, then dip and swirled it in the PCR rxn tube before dip and swirling into labeled microfuge tubes with 50 uL LB media. The microfuge tubes were stored in a 4 degrees Celsius fridge. A DNA gel of the colony PCRs showed that the ligation of nah into the backbone failed - as seen in the accompanying gel picture, our positive control worked (indicating the PCR conditions were fine), but the test colonies all only had 700 bp amplicons. <br />
<br />
Fluorescence tests: Claire found some old SAL2 and mRFP digested with PstI and SpeI already, so she decided to retry the ligation. She did a 40min ligation at room temp, and continued it overnight in the 4degC fridge, which the NEB rep said could increase efficience. However, since she forgot to dephosphorylate SAL2, we will never know if this room temp and overnight in the fridge combo would have been effective because it self ligated. <br />
<br> Site-directed mutagenesis: Swat ran a DpnI digestion of half of the previous day's PCR product, in an attempt to rule out the possibility that DpnI was functioning incorrectly. <br />
<br />
</br><br />
<br><br />
<b>From left lane: Ladder, positive control miniprepped plasmid, colony 1, colony 2, colony 3, colony 4, colony 5, colony 6, colony 7, colony 8, positive control colony:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/3/37/8_31_2012_WM3064_nah_EDIT.jpg/683px-8_31_2012_WM3064_nah_EDIT.jpg"><br />
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<h3>August 1st - 4th</h3><br />
<br><b> August 1st, Wednesday </b></br><br> In the morning, Dylan noted that 5 of the 7 transformations from Tuesday produced plates with colonies. Those that worked were three Anderson series constitutive promoters (with downstream mRFP) in pBBRBB and the versions of our two arsenic reporters with mRFP downstream of mtrB. In the evening, Swati and Dylan prepared overnight cultures from all transformants and JG700, both to set up conjugations to get plasmid into Shewanella and to screen isolated plasmids for successful ligation. We will use these constructs to characterize the activity of the arsenic-sensitive promoter in Shewanella with respect to known constitutive promoter strengths. The two transformations that failed were versions of the salicylate reporter. <br />
After staring at plates, Dylan ran the nah+p29, oriT+p17c, p31c, and nah operon (from p20) digestions on a gel, extracted the fragments of interest, and then dephosphorylated the backbone. The isolated digestion products were used later used by Steven, who set up two overnight ligations – one to put the nah operon, with a constitutive promoter, on a pSB3C5 backbone with an added oriT sequence, and one to put the nah operon into the MCS of pSB1C3 for subsequent site directed mutagenesis. <br />
While Dylan ran his gel, Caleb miniprepped from the JG700+SAL and S31 cultures. After quantification, SAL plasmid isolated from JG700 was submitted for sequencing in order to confirm successful conjugation. <br />
Meanwhile, Mark decided that he enjoyed making DH5a electrocompetent stocks on Tuesday so much that he had to make more WM3064 electrocompetent stocks today. Starting from subcultures that Dylan had prepared, he prepared the stocks, and was assisted by Danielle. <br />
<br><br />
<b>From left: ladder, oriT+p17c digest (E+X), nah + p29 digest (E+S), p31c digest (E+S), p20 PCR digest (E+S):</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/5/5d/2012_08_01_nahsDigest.jpg/800px-2012_08_01_nahsDigest.jpg"><br />
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</br><br />
<br><b> August 2nd, Thursday </b></br><br> Caleb and Dylan created conjugation plates of p14RFP, p16RFP, and p25-29BB as well as miniprepped from the same plasmids in WM3064 (see: strain list). Dylan, Mark, and Tina then desalted and transformed SAL2 and SALRFP into WM3064 as well as p31c_nah and oriT_nah_p17c into DH5a. Tina and Dylan then quantified the minipreps from earlier in the day. Dylan also digested pBBRBB and p33k with EcoRI &amp; PstI to put lac inducible mtrB into the pBBRBB backbone.<br />
</br> <br> </br><br />
<br />
<br><b> August 3rd, Friday </b></br><br> After running a digestion of p33k, the lac inducible mtrB part from the parts registry, we discovered that the part does not include mtrB! We should have seen a ~3.6kb band on our gel after digesting with EcoRI and PstI, but instead isolated a band ~1.5kb. After checking the sequencing on the parts registry we discovered this was because the part didn't include mtrB, so we won't be able to use it as a positive control for inducible mtrB. <br />
Sequencing of p37k-p41k from WM3064 came back good, meaning we can put these control parts, with RFP downstream of our various reporters, into Shewanella via conjugation. Hence, conjugation was carried out. <br />
<br><br />
<b>Digests of p11 and p33 (E+P):</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/6/69/2012_08_03_p11p33dig.jpg/800px-2012_08_03_p11p33dig.jpg"><br />
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</br> <br> </br><br />
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<br><b> August 4th, Saturday </b></br><br> This week, it was confirmed that RFP was successfully inserted downstream of mtrB in our arsenic reporter in Shewenella. <br />
Site-directed mutagenesis of the nah operon failed. We continued trying to insert the nah operon into the mobile backbone (OriT) to prepare for conjugation into Shewanella. <br />
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<h3>August 5th - 11th</h3><br />
<br><b>August 5th, Sunday </b></br><br> Swati yet again was saddled with a massive number of <a href="https://static.igem.org/mediawiki/2012/3/35/Ezna_Miniprep.pdf">minipreps </a>, since her magic fingers are able to cast yield-increasing spells on minipreps. She miniprepped: SAL2 from WM3064, p37-41k from JG700 (one arsenic reporter with cut sites flanking the RBS, another arsenic reporter without the cut sites, and three different Anderson series promoters with mRFP1 downstream on a pBBRBB backbone), oriT p29nah_p17c from DH5a, and nah_p31c from DH5a. And indeed her yields were impressive, massive, gargantuan! Good job Sorceress Swati. <br />
We will <a href="https://static.igem.org/mediawiki/2012/0/03/Sequencing_Preparation.pdf "> sequence </a> SAL2 and oriT p29nah_p17c to see if we should continue to conjugation, and sequence nah_p31c to see if we can start <a href=" https://static.igem.org/mediawiki/2012/a/a5/Site_Directed_Mutagenesis.pdf "> site-directed mutagenesis </a> to get a biobrick-compatible nah operon on the biobrick backbone. We will sequence or PCR to confirm p37k-p41k's conjugation into Shewanella. <br />
</br> <br> </br><br />
<br />
<br />
<br><b> August 6th, Monday </b></br><br> Caleb and Dylan amplified the two different arsenic promoters (p37k and p38k) and ran them on a gel alongside p14 and p16 (the arsenic reporters without mRFP1 downstream) to confirm successful insertion of RFP downstream of mtrB in our arsenic reporter parts in Shewenella. Band lengths appeared in expected places. Dylan is worried that we will not be able to use PCR to definitely confirm that p39-41k worked, so we submitted these for <a href="https://static.igem.org/mediawiki/2012/0/03/Sequencing_Preparation.pdf "> sequencing </a>. Unfortunately, sequencing failed, perhaps because random gunk from Shewanella added noise to the process. <br />
Tina did a PCR cleanup of the nah operon PCR. Dylan also ran a gel of nah operon PCRs to make sure that there was no mispriming. The gel looked good, so he submitted the PCRs for sequencing. <br />
<br><br />
<b>Successful insertion of RFP downstream of mtrB in arsenic reporter strains in Shewanella:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/2012_08_06_p37p38verify.jpg"><br />
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</br> <br> </br><br />
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<br />
<br><b> August 7th, Tuesday </b></br><br> JG1531 overnight culture didn't grow. We suspect that the plate Dylan picked from is dead. We'll have to go back to the glycerol stocks if we want to play with mtrE. Dylan set up a continuous flow M4 reactor in morning. Caleb started a liquid culture of MR-1 with which to inoculate the reactor. Checked sequencing results of nah stuff. oriT thing was bad. nah_p31c was good. <br />
Caleb and Dylan proceeded with <a href=" https://static.igem.org/mediawiki/2012/a/a5/Site_Directed_Mutagenesis.pdf "> site-directed mutagenesis </a> of nah_p31c, attempting to get rid of the first PstI cutsite in the nah operon. After digestion with DpnI, we purified using the E.Z.N.A. MicroElute kit and quantified. DNA was split into three directions: First, Danielle and Dylan set up another mutagenic PCR using the digestion product as template to get rid of the second internal cut site (we expect lower mutation efficiency because template DNA is not methylated). Second, we transformed DH5a with the mutated plasmid. Third, Danielle and Chie <a href="https://static.igem.org/mediawiki/2012/a/af/Double_Digest.pdf "> digested </a> both the purified – and hopefully mutated – plasmid and un-mutated plasmid with PstI. Dylan ran these digestions on a gel, along with supercoiled plasmid as a control. Unfortunately, the (hopefully) mutated plasmid never showed up on the gel. <br />
In the evening, we didn't observe growth in the MR-1 culture, so Dylan set up another culture just in case Shewanella was dead and not lazy. <br />
<br><br />
<b>Looking for mutagenesis – from left – ladder, unmutated nah, mutated nah, supercoil control:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d9/2012_08_07_mutNAHcheck.jpg/800px-2012_08_07_mutNAHcheck.jpg"><br />
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</br> <br> </br><br />
<br />
<br />
<br><b> August 8th, Wednesday </b></br><br> Caleb and Dylan performed another <a href="https://static.igem.org/mediawiki/2012/a/af/Double_Digest.pdf "> digest </a> to check if the second <a href="https://static.igem.org/mediawiki/2012/a/a5/Site_Directed_Mutagenesis.pdf "> site-directed mutagenesis </a> (that was supposed to get rid of the second PstI internal cut site within the nah operon) worked. Unfortunately, there wasn't enough DNA to see anything when visualized on a gel. They decided to proceed with electroporation into DH5a just in case - however, to use Caleb's terminology, cells exploded - there were probably too many salts in the solution, which causing arcing and a PBBHTTTZZZ! of cells all over the cuvette. <br />
Dylan re-digested p29 nah and oriT p17c to redo a ligation that would allow for conjugation into Shewanella later on. Dylan also re-digested p27 (Anderson series promoter with RFP downstream) and SAL to redo a ligation to create a plasmid with SAL-RFP. <br />
Dylan also submitted several samples for <a href="https://static.igem.org/mediawiki/2012/0/03/Sequencing_Preparation.pdf "> sequencing </a> to make sure that three Anderson promoters with RFP downstream in JG700 and SAL2 (the salicylate reporter without the BAMHI cut site) in WN3064 and JG700 were in the correct sequence. <br />
<br><br />
<b>Seeing if mutagenesis worked; nothing there:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/6/6a/2012_08_08_nothingthere.jpg/800px-2012_08_08_nothingthere.jpg"><br />
<br />
</br> <br> </br><br />
<br />
<br />
<br><b> August 9th, Thursday </b></br><br> Today Dylan chilled with his mom. Well, he tried to. :) Despite the eternal bonds that bind mother and son, plus the 3 billion miles she flew to see him, he still couldn't resist coming in to lab to open packages! And then got sucked into a long discussion of what had to be done for the rest of the day - scientific endeavors taking him away yet again from filial duties. Swati and Claire bonded over dry ice, and Caleb regaled us with tales of dry ice bombs gone awry. <br />
Swati and Caleb miniprepped the first mutagenesis of nah_p31c that had been transformed into DH5a yesterday. They then digested it with PstI-HF to see if the mutation was successful. Unfortunately the mutagenesis failed! We will start over from nah_p31c and lower the annealing temperature at 60degC. The Stratagene kit, which uses PFU ultra, asks for 60degC, but because we are using our own boot-leg protocol with Phusion we did the first try at 65degC, as Phusion usually calls for a higher annealing temperature than the theoretically calculated value. For this second try we will stick to the 60degC suggested by Stratagene and see if we get better results. Also, called NEB to find out if after PCR with Phusion, DpnI will still have activity in the following digestion step in Buffer 4, or if we need to clean up the PCR before digesting with DpnI - they said PCR clean up isn't needed. <br />
We also ran digestions for making the nah operon on a backbone with a mobility gene and the salicylate reporter (w/ BamHI cutsite) with RFP downstream. We cut our mobile backbone, OriT in p17c (pSB3C5), with EcoRI and XbaI, while cutting the nah operon (p29nah) with EcoRI and SpeI. The nah operon with mobility gene must be constructed so that we may conjugate into Shewy and start testing our salicylate reporter. The salicylate reporter and p27a (from the Anderson series), with RFP, were cut with SpeI and PstI. The salicylate/RFP part will be used for troubleshooting the salicylate reporter. Digestion products were run on a gel, extracted, and quantified. Swati then dephosphorylated backbones and ligated both parts. <br />
Sequencing for p39-41k didn't look good, and neither did the salicylate reporter w/BamHI cutsite miniprepped from JG700. The sequencing for salicylate reporter w/ BamHI in WM3064, however, looked good, suggesting that conjugation may not have been as efficient as we had hoped. After a pow-wow we decided not to sequence more colonies, as we are hoping some may be good, and more importantly that if we use qPCR to get quantitative characterization data, we won't need to use the RFP parts. The plates will stay in the fridge as a back-up plan. <br />
In other news: Claire cried because it was difficult to update the notebook with a week's worth of work. Mark's dedication to the notebook is laudable and impressive. Good job team for doing so much! My head can't even comprehend the magnitude of your endeavors. <br />
<br><br />
<b>Unsuccessful mutagenesis (test with Pst1 cutsite):</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/8b/2012_08_09_pstI_cut_site_test.jpg/800px-2012_08_09_pstI_cut_site_test.jpg"><br />
<br><br />
<b>Digestion products from left – ladder, p27, p29nah, oriTp17c, SAL:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/1/1e/2012_08_09_SALRFP_nah17_dig_copy.jpg/534px-2012_08_09_SALRFP_nah17_dig_copy.jpg"><br />
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</br> <br> </br><br />
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<br><b> August 10th, Friday </b></br><br> Caleb miniprepped a plasmid with mtrE from JG1531, and just for kicks, miniprepped from the "exploded cells" from August 8th. Suprisingly, he ended up getting decent yields for both, showing that the electroporation worked despite arcing. Caleb then digested the nah operon of the miniprep with PSTI and NotI to check if the mutagenesis was a success. It was run on a gel alongside a PCR of the Anderson series promoters with RFP downstream and SAL2 from Shewanella (to check if SAL2 and the promoters were sucessfully conjugated after sequencing on Monday failed). Unfortunately, bands did not appear where we expected them to. <br />
Steven and Spencer performed a PCR to get mtrE out of the Gralnick (JG700) plasmid. <br />
<br><br />
<b>From left lane: ladder, p39 PCR, p40 PCR, p41 PCR, nah-p31c (P+N digestion), SAL2 PCR:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/df/2012_08_10_longer_exposure.JPG/800px-2012_08_10_longer_exposure.JPG"><br />
</br> <br> </br><br />
<br />
<br><b> August 11th, Saturday </b></br><br> Spencer and Steven checked the nah operon PstI and NotI digest (because yesterday's gel ran weirdly) and their mtrE PCR from the previous day on a gel. Unfortunately, bands did not appear where we expected them to.<br />
<br><br />
<b>mtrE PCR product (left) and checking mutagenesis again (right):</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/7/7e/2012_08_11_mtrE%2C_digest.JPG/800px-2012_08_11_mtrE%2C_digest.JPG"><br />
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<h3>August 12th - 18th</h3><br />
<br><b> August 12th, Sunday </b></br><br> Weekend overview: It looks like the mtrE primers are actually working to amplify the part out of JG1531. We'll be putting mtrE in p31c, both so that we may submit the novel part to the registry, and to begin site-directed mutagenesis. <br />
We'll use mtrE mutagenesis as something of a control for nah operon mutagenesis (to see if the large size of the plasmid and nah operon is the problem). <br />
It also looks like nah operon mutagenesis hasn't worked. We'll put this on hold, and continue with ligation of p29nah into oriT_p17c once desalting paper arrives. <br />
Also, we'll have to redo things to confirm p39-41, SAL2 in JG700. What was done over the weekend looks weird. <br />
</br> <br> </br><br />
<br />
<br><b> August 13th, Monday </b></br><br> Dylan ran four simultaneous Phusion PCRs with an annealing temperature of 67degC and an extension time of 45sec to amplify mtrE. The gel looked good, with no mispriming and one band ~2.2kb. Claire did PCR clean up of the product, which will eventually be cut and ligated into p31c (see: strain list). <br />
Because the results from the weekend's screenings were confusing, we also ran PCRs of p39-41k (miniprepped from JG700 strains) to confirm whether conjugation into Shewanella was successful. These plasmids contain mRFP under the control of constitutive promoters of varying strengths. After confirmation, we will use these parts to characterize our inducible promoters in Shewanella. <br />
Dylan also started a liquid culture of JG700 + SAL to be inoculated into a continuous flow reactor with M4 media. With this setup, we will begin characterizing our reporter strains, initially in response to salicylate. <br />
Dylan performed a Phusion PCR to amplify mtrE, and ran a gel to check for product. There was a single band so no mispriming was occurring. <br />
Claire performed a double digest of the p14k and p16k with XbaI and PstI HF. This is for the construction of our final biobricks to be submitted to the registry. <br />
<br><br />
<b>PCR amplification of mtrE:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c0/2012_08_13_mtrE%2C_PCR.JPG/536px-2012_08_13_mtrE%2C_PCR.JPG"><br />
<br><br />
<b>p39 and p41 verification:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/f/f1/2012_08_13_p3941confirm_overnight.jpg/520px-2012_08_13_p3941confirm_overnight.jpg"><br />
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</br> <br> </br><br />
<br />
<br><b> August 14th, Tuesday </b></br><br> Steven performed a double digest of mtrE (EcorI &amp; SpeI), Sal (SpeI &amp; PstI), Sal2 (SpeI &amp; PstI), and p26a (SpeI &amp; PstI). <br />
Steven performed a ligation of p29nah + oriTp17c, and also a ligation of p27 digest + Sal. Dylan transformed colonies with the ligation. <br />
</br> <br> </br><br />
<br />
<br><b> August 15th, Wednesday </b></br><br> Dylan ran a gel and Claire performed gel extraction of mRFP to put downstream of Sal reporters. They also checked to see if site-directed mutagenesis was successful for the nah-p31c. Found the digest of mRFP and p26 looked good, but the mutagenesis looked strange. <br />
Dylan performed a taq PCR to confirm the presence of Sal2 in Shewanella. However no band was present in the gel ran afterwards, so perhaps colony PCR is the next best step. <br />
Dylan also ran a gel of the p14k, p16k p31 digests (all cut with XbaI and PstI) for the set-up for forming the biobricks. <br />
Steven performed a colony Phusion PCR of four colonies containing the p4k plasmid to again test for the presence of mtrE. Spencer then did PCR clean up <br />
Spencer performed a double digest of p4kdlg and p27a cutting with EcoRI &amp; SpeI followed by running a gel and extracting the digests. <br />
Spencer picked colonies from the previous ligations of the Sal, Sal2, p31c biobrick plasmids for miniprepping on Thursday. <br />
This morning Dylan noted that we may be getting a detectable basal level of current - around 6mA - in a continuous flow setup. This would make it possible to bypass the addition of mtrE to the salicylate sensing part, and still be able to distinguish Shewanella not detecting salicylate from dead Shewanella. He then added salicylate to the reactor innoculated with our salicylate reporting strain, bringing the concentration to 10uM. Later in the day, he noted that the current had risen to around 9mA, which is promising: our strain may be working! Claire set up four hour digestions with XbaI and PstI to put our arsenic parts, p14 and p16, into the iGEM backbone for submission. <br />
Dylan also transformed a salicylate reporter with mRFP downstream into DH5alpha and WM3064, as well as the nah operon with the p29 promoter in a p17c backbone with an oriT. The second of these could be conjugated into Shewanella if transformed successfully. The first, the salicylate reporter with mRFP downstream, is a fluorescent version of the salicylate reporter. The fluorescent versions of our reporters is an alternative to qPCR to measure the relative expression level of mtrB: if we know what mRFP under the control of a constitutive promotor in Shewanella looks like, we can add arsenic or naphthalene/salicylate to our fluorescent reporter parts until reaching the same level of expression. Then, we can correlate this concentration of arsenic or salicylate to a certain strength of induced expression. <br />
In case the fluorescent SAL reporter was not successfully ligated, we are preparing more mRFP, SAL, and SAL2: - In the morning Claire also cleaned up the digestions of SAL, SAL2 and mtrE PCR. However, due to a silly mistake on her part involving wash buffer and absolute ethanol (absolutely missing, to be precise) we will redo the digestions in order to get more DNA for putting these reporters into the iGEM backbone, and to put mRFP downstream. She then set up four hour digestions with XbaI and PstI to put our arsenic parts, p14 and p16, into the iGEM backbone for submission. - We digested mRFP with SpeI and PstI to make the part with mRFP downstream of mtrB in the salicylate reporters. We have already successfully put mRFP downstream of mtrB in our arsenic reporters, but it did not seem to work in the arsenic reporter. The gel for mRFP looked as expected, with the band for the insert slightly shorter than 900bp. However, on the same gel the the mutagenesis trial of the nah operon, digested with PstI and NotI, showed only one band. If mutagenesis had not worked, we would expect more than two bands, and if it had worked we would still expect two bands. Therefore we are unsure how to interpret this result, which we have seen twice now, but are going to start over with site-directed mutagenesis of the nah operon. We will also visualize the unmutated nah operon, digested with PstI and NotI, on a gel to see if that looks like we expect it to. <br />
Finally, it should be noted with jubilance that Claire was reunited with her umbrella today! The joy in the lab at this event was palpable and will be remembered for years to come.<br />
<br><br />
<b>mRFP digest and additional (failed) attempt at confirming mutagenesis:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d9/2012_08_15_mRFPdigest_mutnahPNDigest.JPG/583px-2012_08_15_mRFPdigest_mutnahPNDigest.JPG"><br />
<br><br />
<b>From left – ladder, failed SAL2, p14, p16, p31digestions:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c5/2012_08_15_sal2fail_p14_16_31_dig.jpg/689px-2012_08_15_sal2fail_p14_16_31_dig.jpg"><br />
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</br> <br> </br><br />
<br />
<br><b> August 16th, Thursday </b></br><br> Dylan performed a miniprep of the Sal, Sal2, and p31c reporters for us to do sequencing for confirmation of successful ligations. These will hopefully be the biobricks we send in. <br />
In the afternoon Dylan performed a dephosphorylation of the p31c (cut with XbaI, PstI) followed by a gel of the mtrE cut with EcoRI &amp; SpeI as well as mRFP cut with SpeI and PstI. <br />
A 4 hour digestion was also performed by Claire to get Sal parts into pSB1C3 for the SAL, SAL2, and p31 parts. Steven then ran a gel of the digest products. <br />
Steven also performed a colony Taq PCR of the Sal2 reporter to confirm its presence in Shewanella. <br />
Today, we continued work to get our engineered reporters into pSB1C3 for submission to the parts registry. In the morning, Dylan miniprepped both versions of our salicylate reporters, along with more pSB1C3 from overnight cultures of the corresponding DH5a strains. Following quantification, he set up digestions of each miniprep with XbaI and PstI. (We decided to cut with XbaI because we discovered an extra base pair between the NotI and XbaI cutsites in the normal BioBrick prefix, an artifact of a previous team's work). Caleb also dephosphorylated previously isolated pSB1C3 backbone to prevent self-ligation, while Steven gel extracted the salicylate reporter inserts and more pSB1C3 backbone (to be quantified Friday morning). <br />
<br><br />
<b>mtrE and mRFP digests:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/86/2012_08_16_mtrEdig_mRFPdig.jpg/557px-2012_08_16_mtrEdig_mRFPdig.jpg"><br />
<br><br />
<b>Digestions of SAL, SAL2, and p31:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/7/7f/2012_08_16_SALSAL2p31dig.jpg/553px-2012_08_16_SALSAL2p31dig.jpg"><br />
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</br> <br> </br><br />
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<br><b> August 17th, Friday </b></br><br> Claire performed a ligation for the construction of the Sal_RFP, and Sal2_RFP plasmids. Transformation was then performed immediately after.<br />
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<h3>August 19th - 25th</h3><br />
<br><b> August 19th, Sunday </b></br><br> In the morning, Dylan came to lab to look at the transformant plates from Saturday. There were colonies on all but one of the plates: It looks like either the ligation to put mtrE into p31c or the subsequent electroporation failed. It is of note that the competent DH5α cells that Dylan used for the mtrE electroporation were from an older stock – one which we'd previously determined to confer lower transformation efficiency. Because we used up all of the newer stock – with markedly better efficiency – we will make more electrocompetent DH5α in the coming week.<br />
Because all other transformations seemed to work, we grew up overnight cultures from single colonies on each plate. We will miniprep from these cultures and screen the minipreps for the correct insert. If confirmed, we will have successfully have put both versions of our arsenic reporters, as well as both versions of our salicylate reporters inside p31c (i.e., pSB1C3), as is required for registry submission. We will also have successfully put mRFP downstream of mtrB on SAL2, which will be used for characterization of the salicylate sensitive promoter in S. oneidensis.<br />
<br><br />
<b>Gel showing failure of nahoriT p17c:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/6/6d/2012_08_19_p29nahoriTp17cfail.jpg/584px-2012_08_19_p29nahoriTp17cfail.jpg"><br />
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</br> <br> </br><br />
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<br><b> August 22th, Wednesday </b></br><br> Conjugating nah into Shewy: Caleb, Tina, and a guest Louis miniprepped 5 plasmids containing nah operons with constituitive promoters from a WM3064 plate and miniprepped one plasmid that we previously confirmed as having the nah operon (without a promoter) to use as a positive control in the future. <br />
Claire performed miniprep of grown cultures of the Sal_RFP and Sal2_RFP to submit for sequencing. <br />
</br> <br> </br><br />
<br />
<br><b> August 24th, Friday </b></br><br>Jim performed the digestion of the nah operon+ p29a and the p31c backbone for ultimate ligation in the creation of the last biobrick. Ligation had to be redone, as sequencing results of last week's ligations looked great except for the nah operon biobrick. <br />
<br />
Conjugating nah into Shewy: <br />
We wanted to accomplish two things: <br />
• Amplify the nah operon with a promoter using primers with appended cut sites. <br />
• Optimize colony PCR for future Cornell iGEMers (past attempts have failed.) <br />
To accomplish both of these objectives, we did Phusion PCRs using plasmids containing nah operons with constituitive promoters as a template and a Phusion colony PCR using the same colonies we miniprepped the plasmid from. Because we expect the miniprep PCRs to work, doing the colony PCRs in parallel will allow us to determine whether our colony PCR technique is working or not. For our colony PCR purposes, we want to be able to screen for colonies while being certain we can go back and miniprep the same PCRed colony. Our colony PCR method is as follows: <br />
NOTE: We later determined there was a cataclysmic flaw in the following method, can you figure it out? (wink wink nudge nudge...) <br />
• Label colonies of interest and correspondingly label microfuge tubes <br />
• Add 50 uL ddH2O to each microfuge tube <br />
• Using pipette tip, dip into colonies of interest then dip into labelled microfuge tubes and swirl around to release cells <br />
• Use the the ddH2O+cell mixture as the DNA template for a PCR - instead of adding plain ddH2O to the PCR to bring it up to volume, add the ddH2O+cell mixture <br />
• Confirm your PCR had desired results with a DNA gel, then use the ddH2O+cell mixture from the corresponding microfuge tube the PCR was done on to inoculate a culture or plate. <br />
</br> <br> </br><br />
<br />
<br><b> August 25th, Saturday </b></br><br> Jim performed an overnight ligation of the nah operon pSB1C3 for transformation on Sunday. <br />
Conjugating nah into Shewy: <br />
Caleb, Tina, and a guest Louis ran a gel of the miniprep and colony parallel Phusion PCRs. All of the miniprep PCRs worked, but only a couple colony PCRs worked. We believe this is due to not having added enough cells to the colony PCR tube because some of the colonies were very tiny. <br />
<br><br />
<b>Top: Positive control from colony, colony test 1, colony test 2, colony test 3, colony test 4, colony test 5, miniprep 5, Benchtop 1kb; Bottom: Benchtop 1kb, miniprep 1, miniprep 2, miniprep 3, miniprep 4, positive control miniprep:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c9/2012_8_25_Checking_for_nah_product_from_colony_vs_miniprep_PCRs.jpg/489px-2012_8_25_Checking_for_nah_product_from_colony_vs_miniprep_PCRs.jpg"><br />
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<h3>August 26th - 31st</h3><br />
<br><b> August 26th, Sunday </b></br><br> Conjugating nah into Shewy: <br />
Caleb miniprepped two plasmids - one contained the nah operon preceded with a constitutive promoter and the other is to become a backbone containing the origin of transfer required for conjugation and a gene conveying chloramphenicol resistance. The purified plasmids were digested to extract the nah operon and to cut open the backbone - these digests were then run on a gel. Tina gel extracted the backbone then accidentally threw away the rest of the gel that contained the nah operon fragments. <br />
<br> Site-directed mutagenesis: Swati ran a mutagenic PCR of nah_p31c, then digested with DpnI to remove template DNA and column purified the resultant DNA. </br><br />
</br> <br> </br><br />
<br />
<br />
<br><b> August 27th, Monday </b></br><br> Conjugating nah into Shewy: <br />
Caleb gel extracted the nah operon containing fragments from yesterday - despite having been in the garbage overnight, the gel extraction worked! Caleb and Tina began a 24 hour 16 degrees Celsius ligation using molar rations of nah to backbone of 3:1 and 1:1. <br />
Fluorescence tests: Claire started a JG700 + SAL2 culture and tried to do a PCR of SAL2_mRFP with sequencing primers to see if the band produced would correspond to the length of SAL2 plus the length of mRFP. Unfortunately, due to a mishap involving p10s vs. p2s and not checking what volume her pipette was set to, she ended up with a 70uL total volume and PCR failed. <br />
<br> Site-directed mutagenesis: Swati transformed the (hopefully) mutated DNA into DH5a. </br><br />
</br> <br> </br><br />
<br />
<br><b> August 28th, Tuesday </b></br><br> Conjugating nah into Shewy: <br />
Caleb and Tina de-salted the 3:1 and 1:1 ligations, then transformed them into a DAP-requiring conjugation strain WM3064 via electroporation. The electroporated cells were plated on DAP + Cm plates. <br />
Fluorescence tests: JG700 once again takes a long time to grow, so it wasn’t until the evening that Claire miniprepped SAL2 from JG700. Tomorrow we will run another PCR and see if we have SAL2_mRFP, and SAL2 in JG700. <br />
<br> Site-directed mutagenesis: Swati's transformation did not work. After checking the PCR product and a PstI digest thereof on a gel, she discovered that the PCR was not working with high enough efficiency to produce a band on a gel. Upon consulting with Didi, our trusty post-doctoral adviser, she then restarted the mutagenic process with a PCR; this time, with a lower primer concentration and lower denaturation and annealing times. </br><br />
</br> <br> </br><br />
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<br><b> August 29th, Wednesday </b></br><br> Conjugating nah into Shewy: <br />
Caleb checked the transformation plates after ~14 hours in a 37 degrees Celsius incubator and noticed colonies, but the colonies were barely visible indicating very slow growth of the cells. <br />
Fluorescence tests: Set up a Taq PCR of SAL_mRFP, SAL2_mRFP and SAL2, with an extension time of 4.5 minutes and annealing temperature of 68degC. SAL2_mRFP has a band corresponding only to the length of SAL2, without mRFP, so we need to redo this ligation. However, the band from SAL_mRFP was the correct length (a little more than 4kb), so this ligation was successful and we can conjugate into Shewenella. Finally, the JG700 miniprep is the correct length for SAL2 (~3.8kb), so SAL2 has been successfully conjugated into Shewenella! <br />
<br> Site-directed mutagenesis: Swati digested her second mutagenic PCR with DpnI, column purified, and transformed into DH5a. </br><br />
</br> <br> </br><br />
<br />
<br><b> August 30th, Thursday </b></br><br> Conjugating nah into Shewy: <br />
Tina checked the growth of yesterday's colonies and tried doing a colony PCR of each colony and a PCR of a positive control (same product, same primes, but in a different purified plasmid). The primers were designed to sit on the backbone and face into the oriT region and the site where we wanted to insert the nah operon. A PCR amplicon indicating successful ligation of the nah operon into the backbone would be approximately 10-11kb, while a failed ligation would yield a ~700 bp amplicon. Caleb later ran a DNA gel of the colony PCRs and nothing but smears showed up in every lane of the gel except the positive control which didn't even have a smear (Caleb was so disappointed that he didn't post the picture). Since the positive control didn't show up, we concluded the PCR failed. Caleb started overnight cultures of the remaining cells+ddH2O used for the colony PCR. <br />
<br> Site-directed mutagenesis: Alas, Swati's second attempt at mutagenesis was yet again fruitless. Concerned that the template may have been causing the problem, she performed a NotI digest and ran it on a gel alongside the latest mutagenic PCR. The template was fine; the PCR was, again, invisible. Swati grew extremely puzzled as to how her purified PCR product could have significant concentration upon quantification but yield no visible bands on a gel. She decided to try a third time, this time with longer denaturation and annealing times, but still the same concentration of primers.<br />
<br><br />
<b>From left lane: Ladder, SAL2_mRFP (1), SAL2_mRFP (2), SAL2:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/f/fc/2012_08_30_gel.jpg/800px-2012_08_30_gel.jpg"><br />
</br><br />
</br> <br> </br><br />
<br />
<br><b> August 31st, Friday </b></br><br> Conjugating nah into Shewy: Caleb noticed the overnight cultures didn't grow and concluded the cells must have died due to the cells being stored in an extremely hypotonic solution (ddH2O). He re-did colony PCRs of the colonies, a colony with a positive control, and a positive control of purified plasmid, but modified the colony PCR protocol. Instead of using the cells + ddH2O as template DNA, Caleb dipped the pipette tip into the colony, then dip and swirled it in the PCR rxn tube before dip and swirling into labeled microfuge tubes with 50 uL LB media. The microfuge tubes were stored in a 4 degrees Celsius fridge. A DNA gel of the colony PCRs showed that the ligation of nah into the backbone failed - as seen in the accompanying gel picture, our positive control worked (indicating the PCR conditions were fine), but the test colonies all only had 700 bp amplicons. <br />
<br />
Fluorescence tests: Claire found some old SAL2 and mRFP digested with PstI and SpeI already, so she decided to retry the ligation. She did a 40min ligation at room temp, and continued it overnight in the 4degC fridge, which the NEB rep said could increase efficience. However, since she forgot to dephosphorylate SAL2, we will never know if this room temp and overnight in the fridge combo would have been effective because it self ligated. <br />
<br> Site-directed mutagenesis: Swat ran a DpnI digestion of half of the previous day's PCR product, in an attempt to rule out the possibility that DpnI was functioning incorrectly. <br />
</br><br />
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<b>From left lane: Ladder, positive control miniprepped plasmid, colony 1, colony 2, colony 3, colony 4, colony 5, colony 6, colony 7, colony 8, positive control colony:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/3/37/8_31_2012_WM3064_nah_EDIT.jpg/683px-8_31_2012_WM3064_nah_EDIT.jpg"><br />
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<h3>August 1st - 4th</h3><br />
It was confirmed that RFP was successfully inserted downstream of mtrB in our arsenic reporter parts in Shewanella. Site-directed mutagenesis of the nah operon failed. We are still trying to insert the nah operon into the mobility backbone (OriT) to prepare for conjugation into Shewanella later on.<br />
<div class="panel" style="background:white;margin-top: 20px;"><br />
<h6>Daily Details:</h6><br />
<br><b> August 1st, Wednesday </b></br><br> In the morning, Dylan noted that 5 of the 7 transformations from Tuesday produced plates with colonies. Those that worked were three Anderson series constitutive promoters (with downstream mRFP) in pBBRBB and the versions of our two arsenic reporters with mRFP downstream of mtrB. In the evening, Swati and Dylan prepared overnight cultures from all transformants and JG700, both to set up conjugations to get plasmid into Shewanella and to screen isolated plasmids for successful ligation. We will use these constructs to characterize the activity of the arsenic-sensitive promoter in Shewanella with respect to known constitutive promoter strengths. The two transformations that failed were versions of the salicylate reporter. <br />
After staring at plates, Dylan ran the nah+p29, oriT+p17c, p31c, and nah operon (from p20) digestions on a gel, extracted the fragments of interest, and then dephosphorylated the backbone. The isolated digestion products were used later used by Steven, who set up two overnight ligations – one to put the nah operon, with a constitutive promoter, on a pSB3C5 backbone with an added oriT sequence, and one to put the nah operon into the MCS of pSB1C3 for subsequent site directed mutagenesis. <br />
While Dylan ran his gel, Caleb miniprepped from the JG700+SAL and S31 cultures. After quantification, SAL plasmid isolated from JG700 was submitted for sequencing in order to confirm successful conjugation. <br />
Meanwhile, Mark decided that he enjoyed making DH5a electrocompetent stocks on Tuesday so much that he had to make more WM3064 electrocompetent stocks today. Starting from subcultures that Dylan had prepared, he prepared the stocks, and was assisted by Danielle. <br />
<br><br />
<b>From left: ladder, oriT+p17c digest (E+X), nah + p29 digest (E+S), p31c digest (E+S), p20 PCR digest (E+S):</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/5/5d/2012_08_01_nahsDigest.jpg/800px-2012_08_01_nahsDigest.jpg"><br />
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</br><br />
<br><b> August 2nd, Thursday </b></br><br> Caleb and Dylan created conjugation plates of p14RFP, p16RFP, and p25-29BB as well as miniprepped from the same plasmids in WM3064 (see: strain list). Dylan, Mark, and Tina then desalted and transformed SAL2 and SALRFP into WM3064 as well as p31c_nah and oriT_nah_p17c into DH5a. Tina and Dylan then quantified the minipreps from earlier in the day. Dylan also digested pBBRBB and p33k with EcoRI &amp; PstI to put lac inducible mtrB into the pBBRBB backbone.<br />
</br> <br> </br><br />
<br />
<br><b> August 3rd, Friday </b></br><br> After running a digestion of p33k, the lac inducible mtrB part from the parts registry, we discovered that the part does not include mtrB! We should have seen a ~3.6kb band on our gel after digesting with EcoRI and PstI, but instead isolated a band ~1.5kb. After checking the sequencing on the parts registry we discovered this was because the part didn't include mtrB, so we won't be able to use it as a positive control for inducible mtrB. <br />
Sequencing of p37k-p41k from WM3064 came back good, meaning we can put these control parts, with RFP downstream of our various reporters, into Shewanella via conjugation. Hence, conjugation was carried out. <br />
<br><br />
<b>Digests of p11 and p33 (E+P):</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/6/69/2012_08_03_p11p33dig.jpg/800px-2012_08_03_p11p33dig.jpg"><br />
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</br> <br> </br><br />
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<br><b> August 4th, Saturday </b></br><br> This week, it was confirmed that RFP was successfully inserted downstream of mtrB in our arsenic reporter in Shewenella. <br />
Site-directed mutagenesis of the nah operon failed. We continued trying to insert the nah operon into the mobile backbone (OriT) to prepare for conjugation into Shewanella. <br />
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<h3>August 5th - 11th</h3><br />
This week, we continued our site directed mutagenesis of nah_p31c and the addition of RFP to our SAL reporters. Sequencing of the arsenic reporter has been unsuccessful so far. We also decided that we need more quantitative data to characterize our parts.<br />
<div class="panel" style="background:white;margin-top: 20px;"><br />
<h6>Daily Details:</h6><br />
<br><b>August 5th, Sunday </b></br><br> Swati yet again was saddled with a massive number of <a href="https://static.igem.org/mediawiki/2012/3/35/Ezna_Miniprep.pdf">minipreps </a>, since her magic fingers are able to cast yield-increasing spells on minipreps. She miniprepped: SAL2 from WM3064, p37-41k from JG700 (one arsenic reporter with cut sites flanking the RBS, another arsenic reporter without the cut sites, and three different Anderson series promoters with mRFP1 downstream on a pBBRBB backbone), oriT p29nah_p17c from DH5a, and nah_p31c from DH5a. And indeed her yields were impressive, massive, gargantuan! Good job Sorceress Swati. <br />
We will <a href="https://static.igem.org/mediawiki/2012/0/03/Sequencing_Preparation.pdf "> sequence </a> SAL2 and oriT p29nah_p17c to see if we should continue to conjugation, and sequence nah_p31c to see if we can start <a href=" https://static.igem.org/mediawiki/2012/a/a5/Site_Directed_Mutagenesis.pdf "> site-directed mutagenesis </a> to get a biobrick-compatible nah operon on the biobrick backbone. We will sequence or PCR to confirm p37k-p41k's conjugation into Shewanella. <br />
</br> <br> </br><br />
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<br />
<br><b> August 6th, Monday </b></br><br> Caleb and Dylan amplified the two different arsenic promoters (p37k and p38k) and ran them on a gel alongside p14 and p16 (the arsenic reporters without mRFP1 downstream) to confirm successful insertion of RFP downstream of mtrB in our arsenic reporter parts in Shewenella. Band lengths appeared in expected places. Dylan is worried that we will not be able to use PCR to definitely confirm that p39-41k worked, so we submitted these for <a href="https://static.igem.org/mediawiki/2012/0/03/Sequencing_Preparation.pdf "> sequencing </a>. Unfortunately, sequencing failed, perhaps because random gunk from Shewanella added noise to the process. <br />
Tina did a PCR cleanup of the nah operon PCR. Dylan also ran a gel of nah operon PCRs to make sure that there was no mispriming. The gel looked good, so he submitted the PCRs for sequencing. <br />
<br><br />
<b>Successful insertion of RFP downstream of mtrB in arsenic reporter strains in Shewanella:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/2012_08_06_p37p38verify.jpg"><br />
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</br> <br> </br><br />
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<br />
<br><b> August 7th, Tuesday </b></br><br> JG1531 overnight culture didn't grow. We suspect that the plate Dylan picked from is dead. We'll have to go back to the glycerol stocks if we want to play with mtrE. Dylan set up a continuous flow M4 reactor in morning. Caleb started a liquid culture of MR-1 with which to inoculate the reactor. Checked sequencing results of nah stuff. oriT thing was bad. nah_p31c was good. <br />
Caleb and Dylan proceeded with <a href=" https://static.igem.org/mediawiki/2012/a/a5/Site_Directed_Mutagenesis.pdf "> site-directed mutagenesis </a> of nah_p31c, attempting to get rid of the first PstI cutsite in the nah operon. After digestion with DpnI, we purified using the E.Z.N.A. MicroElute kit and quantified. DNA was split into three directions: First, Danielle and Dylan set up another mutagenic PCR using the digestion product as template to get rid of the second internal cut site (we expect lower mutation efficiency because template DNA is not methylated). Second, we transformed DH5a with the mutated plasmid. Third, Danielle and Chie <a href="https://static.igem.org/mediawiki/2012/a/af/Double_Digest.pdf "> digested </a> both the purified – and hopefully mutated – plasmid and un-mutated plasmid with PstI. Dylan ran these digestions on a gel, along with supercoiled plasmid as a control. Unfortunately, the (hopefully) mutated plasmid never showed up on the gel. <br />
In the evening, we didn't observe growth in the MR-1 culture, so Dylan set up another culture just in case Shewanella was dead and not lazy. <br />
<br><br />
<b>Looking for mutagenesis – from left – ladder, unmutated nah, mutated nah, supercoil control:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d9/2012_08_07_mutNAHcheck.jpg/800px-2012_08_07_mutNAHcheck.jpg"><br />
<br />
</br> <br> </br><br />
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<br />
<br><b> August 8th, Wednesday </b></br><br> Caleb and Dylan performed another <a href="https://static.igem.org/mediawiki/2012/a/af/Double_Digest.pdf "> digest </a> to check if the second <a href="https://static.igem.org/mediawiki/2012/a/a5/Site_Directed_Mutagenesis.pdf "> site-directed mutagenesis </a> (that was supposed to get rid of the second PstI internal cut site within the nah operon) worked. Unfortunately, there wasn't enough DNA to see anything when visualized on a gel. They decided to proceed with electroporation into DH5a just in case - however, to use Caleb's terminology, cells exploded - there were probably too many salts in the solution, which causing arcing and a PBBHTTTZZZ! of cells all over the cuvette. <br />
Dylan re-digested p29 nah and oriT p17c to redo a ligation that would allow for conjugation into Shewanella later on. Dylan also re-digested p27 (Anderson series promoter with RFP downstream) and SAL to redo a ligation to create a plasmid with SAL-RFP. <br />
Dylan also submitted several samples for <a href="https://static.igem.org/mediawiki/2012/0/03/Sequencing_Preparation.pdf "> sequencing </a> to make sure that three Anderson promoters with RFP downstream in JG700 and SAL2 (the salicylate reporter without the BAMHI cut site) in WN3064 and JG700 were in the correct sequence. <br />
<br><br />
<b>Seeing if mutagenesis worked; nothing there:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/6/6a/2012_08_08_nothingthere.jpg/800px-2012_08_08_nothingthere.jpg"><br />
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</br> <br> </br><br />
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<br><b> August 9th, Thursday </b></br><br> Today Dylan chilled with his mom. Well, he tried to. :) Despite the eternal bonds that bind mother and son, plus the 3 billion miles she flew to see him, he still couldn't resist coming in to lab to open packages! And then got sucked into a long discussion of what had to be done for the rest of the day - scientific endeavors taking him away yet again from filial duties. Swati and Claire bonded over dry ice, and Caleb regaled us with tales of dry ice bombs gone awry. <br />
Swati and Caleb miniprepped the first mutagenesis of nah_p31c that had been transformed into DH5a yesterday. They then digested it with PstI-HF to see if the mutation was successful. Unfortunately the mutagenesis failed! We will start over from nah_p31c and lower the annealing temperature at 60degC. The Stratagene kit, which uses PFU ultra, asks for 60degC, but because we are using our own boot-leg protocol with Phusion we did the first try at 65degC, as Phusion usually calls for a higher annealing temperature than the theoretically calculated value. For this second try we will stick to the 60degC suggested by Stratagene and see if we get better results. Also, called NEB to find out if after PCR with Phusion, DpnI will still have activity in the following digestion step in Buffer 4, or if we need to clean up the PCR before digesting with DpnI - they said PCR clean up isn't needed. <br />
We also ran digestions for making the nah operon on a backbone with a mobility gene and the salicylate reporter (w/ BamHI cutsite) with RFP downstream. We cut our mobile backbone, OriT in p17c (pSB3C5), with EcoRI and XbaI, while cutting the nah operon (p29nah) with EcoRI and SpeI. The nah operon with mobility gene must be constructed so that we may conjugate into Shewy and start testing our salicylate reporter. The salicylate reporter and p27a (from the Anderson series), with RFP, were cut with SpeI and PstI. The salicylate/RFP part will be used for troubleshooting the salicylate reporter. Digestion products were run on a gel, extracted, and quantified. Swati then dephosphorylated backbones and ligated both parts. <br />
Sequencing for p39-41k didn't look good, and neither did the salicylate reporter w/BamHI cutsite miniprepped from JG700. The sequencing for salicylate reporter w/ BamHI in WM3064, however, looked good, suggesting that conjugation may not have been as efficient as we had hoped. After a pow-wow we decided not to sequence more colonies, as we are hoping some may be good, and more importantly that if we use qPCR to get quantitative characterization data, we won't need to use the RFP parts. The plates will stay in the fridge as a back-up plan. <br />
In other news: Claire cried because it was difficult to update the notebook with a week's worth of work. Mark's dedication to the notebook is laudable and impressive. Good job team for doing so much! My head can't even comprehend the magnitude of your endeavors. <br />
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<b>Unsuccessful mutagenesis (test with Pst1 cutsite):</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/8b/2012_08_09_pstI_cut_site_test.jpg/800px-2012_08_09_pstI_cut_site_test.jpg"><br />
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<b>Digestion products from left – ladder, p27, p29nah, oriTp17c, SAL:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/1/1e/2012_08_09_SALRFP_nah17_dig_copy.jpg/534px-2012_08_09_SALRFP_nah17_dig_copy.jpg"><br />
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<br><b> August 10th, Friday </b></br><br> Caleb miniprepped a plasmid with mtrE from JG1531, and just for kicks, miniprepped from the "exploded cells" from August 8th. Suprisingly, he ended up getting decent yields for both, showing that the electroporation worked despite arcing. Caleb then digested the nah operon of the miniprep with PSTI and NotI to check if the mutagenesis was a success. It was run on a gel alongside a PCR of the Anderson series promoters with RFP downstream and SAL2 from Shewanella (to check if SAL2 and the promoters were sucessfully conjugated after sequencing on Monday failed). Unfortunately, bands did not appear where we expected them to. <br />
Steven and Spencer performed a PCR to get mtrE out of the Gralnick (JG700) plasmid. <br />
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<b>From left lane: ladder, p39 PCR, p40 PCR, p41 PCR, nah-p31c (P+N digestion), SAL2 PCR:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/df/2012_08_10_longer_exposure.JPG/800px-2012_08_10_longer_exposure.JPG"><br />
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<br><b> August 11th, Saturday </b></br><br> Spencer and Steven checked the nah operon PstI and NotI digest (because yesterday's gel ran weirdly) and their mtrE PCR from the previous day on a gel. Unfortunately, bands did not appear where we expected them to.<br />
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<b>mtrE PCR product (left) and checking mutagenesis again (right):</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/7/7e/2012_08_11_mtrE%2C_digest.JPG/800px-2012_08_11_mtrE%2C_digest.JPG"><br />
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<h3>August 12th - 18th</h3><br />
This week, we: <br />
1)Performed the neccessary work for getting our reporters into the pSB1C3 backbone required for submission. This included the neccessary digestions of our reporters out and ligations into the submission plasmid. Confirmation of successful transformation and ligation will be performed in the following weeks. <br />
2)Confirmed successful transformation of Shewanella with our arsenic and salicylate reporters via colony PCR. <br />
3)Performed the neccessary digestions and ligations to have mRFP downstream of our reporter system so as to do an additional fluorescent testing of the reporters to serve a second form of confirmation of increased transcription in the presence of our toxins. <br />
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<h6>Daily Details:</h6><br />
<br><b> August 12th, Sunday </b></br><br> Weekend overview: It looks like the mtrE primers are actually working to amplify the part out of JG1531. We'll be putting mtrE in p31c, both so that we may submit the novel part to the registry, and to begin site-directed mutagenesis. <br />
We'll use mtrE mutagenesis as something of a control for nah operon mutagenesis (to see if the large size of the plasmid and nah operon is the problem). <br />
It also looks like nah operon mutagenesis hasn't worked. We'll put this on hold, and continue with ligation of p29nah into oriT_p17c once desalting paper arrives. <br />
Also, we'll have to redo things to confirm p39-41, SAL2 in JG700. What was done over the weekend looks weird. <br />
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<br><b> August 13th, Monday </b></br><br> Dylan ran four simultaneous Phusion PCRs with an annealing temperature of 67degC and an extension time of 45sec to amplify mtrE. The gel looked good, with no mispriming and one band ~2.2kb. Claire did PCR clean up of the product, which will eventually be cut and ligated into p31c (see: strain list). <br />
Because the results from the weekend's screenings were confusing, we also ran PCRs of p39-41k (miniprepped from JG700 strains) to confirm whether conjugation into Shewanella was successful. These plasmids contain mRFP under the control of constitutive promoters of varying strengths. After confirmation, we will use these parts to characterize our inducible promoters in Shewanella. <br />
Dylan also started a liquid culture of JG700 + SAL to be inoculated into a continuous flow reactor with M4 media. With this setup, we will begin characterizing our reporter strains, initially in response to salicylate. <br />
Dylan performed a Phusion PCR to amplify mtrE, and ran a gel to check for product. There was a single band so no mispriming was occurring. <br />
Claire performed a double digest of the p14k and p16k with XbaI and PstI HF. This is for the construction of our final biobricks to be submitted to the registry. <br />
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<b>PCR amplification of mtrE:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c0/2012_08_13_mtrE%2C_PCR.JPG/536px-2012_08_13_mtrE%2C_PCR.JPG"><br />
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<b>p39 and p41 verification:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/f/f1/2012_08_13_p3941confirm_overnight.jpg/520px-2012_08_13_p3941confirm_overnight.jpg"><br />
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<br><b> August 14th, Tuesday </b></br><br> Steven performed a double digest of mtrE (EcorI &amp; SpeI), Sal (SpeI &amp; PstI), Sal2 (SpeI &amp; PstI), and p26a (SpeI &amp; PstI). <br />
Steven performed a ligation of p29nah + oriTp17c, and also a ligation of p27 digest + Sal. Dylan transformed colonies with the ligation. <br />
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<br><b> August 15th, Wednesday </b></br><br> Dylan ran a gel and Claire performed gel extraction of mRFP to put downstream of Sal reporters. They also checked to see if site-directed mutagenesis was successful for the nah-p31c. Found the digest of mRFP and p26 looked good, but the mutagenesis looked strange. <br />
Dylan performed a taq PCR to confirm the presence of Sal2 in Shewanella. However no band was present in the gel ran afterwards, so perhaps colony PCR is the next best step. <br />
Dylan also ran a gel of the p14k, p16k p31 digests (all cut with XbaI and PstI) for the set-up for forming the biobricks. <br />
Steven performed a colony Phusion PCR of four colonies containing the p4k plasmid to again test for the presence of mtrE. Spencer then did PCR clean up <br />
Spencer performed a double digest of p4kdlg and p27a cutting with EcoRI &amp; SpeI followed by running a gel and extracting the digests. <br />
Spencer picked colonies from the previous ligations of the Sal, Sal2, p31c biobrick plasmids for miniprepping on Thursday. <br />
This morning Dylan noted that we may be getting a detectable basal level of current - around 6mA - in a continuous flow setup. This would make it possible to bypass the addition of mtrE to the salicylate sensing part, and still be able to distinguish Shewanella not detecting salicylate from dead Shewanella. He then added salicylate to the reactor innoculated with our salicylate reporting strain, bringing the concentration to 10uM. Later in the day, he noted that the current had risen to around 9mA, which is promising: our strain may be working! Claire set up four hour digestions with XbaI and PstI to put our arsenic parts, p14 and p16, into the iGEM backbone for submission. <br />
Dylan also transformed a salicylate reporter with mRFP downstream into DH5alpha and WM3064, as well as the nah operon with the p29 promoter in a p17c backbone with an oriT. The second of these could be conjugated into Shewanella if transformed successfully. The first, the salicylate reporter with mRFP downstream, is a fluorescent version of the salicylate reporter. The fluorescent versions of our reporters is an alternative to qPCR to measure the relative expression level of mtrB: if we know what mRFP under the control of a constitutive promotor in Shewanella looks like, we can add arsenic or naphthalene/salicylate to our fluorescent reporter parts until reaching the same level of expression. Then, we can correlate this concentration of arsenic or salicylate to a certain strength of induced expression. <br />
In case the fluorescent SAL reporter was not successfully ligated, we are preparing more mRFP, SAL, and SAL2: - In the morning Claire also cleaned up the digestions of SAL, SAL2 and mtrE PCR. However, due to a silly mistake on her part involving wash buffer and absolute ethanol (absolutely missing, to be precise) we will redo the digestions in order to get more DNA for putting these reporters into the iGEM backbone, and to put mRFP downstream. She then set up four hour digestions with XbaI and PstI to put our arsenic parts, p14 and p16, into the iGEM backbone for submission. - We digested mRFP with SpeI and PstI to make the part with mRFP downstream of mtrB in the salicylate reporters. We have already successfully put mRFP downstream of mtrB in our arsenic reporters, but it did not seem to work in the arsenic reporter. The gel for mRFP looked as expected, with the band for the insert slightly shorter than 900bp. However, on the same gel the the mutagenesis trial of the nah operon, digested with PstI and NotI, showed only one band. If mutagenesis had not worked, we would expect more than two bands, and if it had worked we would still expect two bands. Therefore we are unsure how to interpret this result, which we have seen twice now, but are going to start over with site-directed mutagenesis of the nah operon. We will also visualize the unmutated nah operon, digested with PstI and NotI, on a gel to see if that looks like we expect it to. <br />
Finally, it should be noted with jubilance that Claire was reunited with her umbrella today! The joy in the lab at this event was palpable and will be remembered for years to come.<br />
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<b>mRFP digest and additional (failed) attempt at confirming mutagenesis:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d9/2012_08_15_mRFPdigest_mutnahPNDigest.JPG/583px-2012_08_15_mRFPdigest_mutnahPNDigest.JPG"><br />
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<b>From left – ladder, failed SAL2, p14, p16, p31digestions:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c5/2012_08_15_sal2fail_p14_16_31_dig.jpg/689px-2012_08_15_sal2fail_p14_16_31_dig.jpg"><br />
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<br><b> August 16th, Thursday </b></br><br> Dylan performed a miniprep of the Sal, Sal2, and p31c reporters for us to do sequencing for confirmation of successful ligations. These will hopefully be the biobricks we send in. <br />
In the afternoon Dylan performed a dephosphorylation of the p31c (cut with XbaI, PstI) followed by a gel of the mtrE cut with EcoRI &amp; SpeI as well as mRFP cut with SpeI and PstI. <br />
A 4 hour digestion was also performed by Claire to get Sal parts into pSB1C3 for the SAL, SAL2, and p31 parts. Steven then ran a gel of the digest products. <br />
Steven also performed a colony Taq PCR of the Sal2 reporter to confirm its presence in Shewanella. <br />
Today, we continued work to get our engineered reporters into pSB1C3 for submission to the parts registry. In the morning, Dylan miniprepped both versions of our salicylate reporters, along with more pSB1C3 from overnight cultures of the corresponding DH5a strains. Following quantification, he set up digestions of each miniprep with XbaI and PstI. (We decided to cut with XbaI because we discovered an extra base pair between the NotI and XbaI cutsites in the normal BioBrick prefix, an artifact of a previous team's work). Caleb also dephosphorylated previously isolated pSB1C3 backbone to prevent self-ligation, while Steven gel extracted the salicylate reporter inserts and more pSB1C3 backbone (to be quantified Friday morning). <br />
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<b>mtrE and mRFP digests:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/86/2012_08_16_mtrEdig_mRFPdig.jpg/557px-2012_08_16_mtrEdig_mRFPdig.jpg"><br />
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<b>Digestions of SAL, SAL2, and p31:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/7/7f/2012_08_16_SALSAL2p31dig.jpg/553px-2012_08_16_SALSAL2p31dig.jpg"><br />
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<br><b> August 17th, Friday </b></br><br> Claire performed a ligation for the construction of the Sal_RFP, and Sal2_RFP plasmids. Transformation was then performed immediately after.<br />
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<h3>August 19th - 25th</h3><br />
Conjugating nah into Shewy: <br />
Colony PCR was done to 1) make sure the nah operon was in the cells 2) troubleshoot the colony PCR protocol After imaging, we discovered that the colonies did contain the nah operon, and that our colony PCR protocol was good.<br />
<div class="panel" style="background:white;margin-top: 20px;"><br />
<h6>Daily Details:</h6><br />
<br><b> August 19th, Sunday </b></br><br> In the morning, Dylan came to lab to look at the transformant plates from Saturday. There were colonies on all but one of the plates: It looks like either the ligation to put mtrE into p31c or the subsequent electroporation failed. It is of note that the competent DH5α cells that Dylan used for the mtrE electroporation were from an older stock – one which we'd previously determined to confer lower transformation efficiency. Because we used up all of the newer stock – with markedly better efficiency – we will make more electrocompetent DH5α in the coming week.<br />
Because all other transformations seemed to work, we grew up overnight cultures from single colonies on each plate. We will miniprep from these cultures and screen the minipreps for the correct insert. If confirmed, we will have successfully have put both versions of our arsenic reporters, as well as both versions of our salicylate reporters inside p31c (i.e., pSB1C3), as is required for registry submission. We will also have successfully put mRFP downstream of mtrB on SAL2, which will be used for characterization of the salicylate sensitive promoter in S. oneidensis.<br />
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<b>Gel showing failure of nahoriT p17c:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/6/6d/2012_08_19_p29nahoriTp17cfail.jpg/584px-2012_08_19_p29nahoriTp17cfail.jpg"><br />
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<br><b> August 22th, Wednesday </b></br><br> Conjugating nah into Shewy: Caleb, Tina, and a guest Louis miniprepped 5 plasmids containing nah operons with constituitive promoters from a WM3064 plate and miniprepped one plasmid that we previously confirmed as having the nah operon (without a promoter) to use as a positive control in the future. <br />
Claire performed miniprep of grown cultures of the Sal_RFP and Sal2_RFP to submit for sequencing. <br />
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<br><b> August 24th, Friday </b></br><br>Jim performed the digestion of the nah operon+ p29a and the p31c backbone for ultimate ligation in the creation of the last biobrick. Ligation had to be redone, as sequencing results of last week's ligations looked great except for the nah operon biobrick. <br />
<br />
Conjugating nah into Shewy: <br />
We wanted to accomplish two things: <br />
• Amplify the nah operon with a promoter using primers with appended cut sites. <br />
• Optimize colony PCR for future Cornell iGEMers (past attempts have failed.) <br />
To accomplish both of these objectives, we did Phusion PCRs using plasmids containing nah operons with constituitive promoters as a template and a Phusion colony PCR using the same colonies we miniprepped the plasmid from. Because we expect the miniprep PCRs to work, doing the colony PCRs in parallel will allow us to determine whether our colony PCR technique is working or not. For our colony PCR purposes, we want to be able to screen for colonies while being certain we can go back and miniprep the same PCRed colony. Our colony PCR method is as follows: <br />
NOTE: We later determined there was a cataclysmic flaw in the following method, can you figure it out? (wink wink nudge nudge...) <br />
• Label colonies of interest and correspondingly label microfuge tubes <br />
• Add 50 uL ddH2O to each microfuge tube <br />
• Using pipette tip, dip into colonies of interest then dip into labelled microfuge tubes and swirl around to release cells <br />
• Use the the ddH2O+cell mixture as the DNA template for a PCR - instead of adding plain ddH2O to the PCR to bring it up to volume, add the ddH2O+cell mixture <br />
• Confirm your PCR had desired results with a DNA gel, then use the ddH2O+cell mixture from the corresponding microfuge tube the PCR was done on to inoculate a culture or plate. <br />
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<br><b> August 25th, Saturday </b></br><br> Jim performed an overnight ligation of the nah operon pSB1C3 for transformation on Sunday. <br />
Conjugating nah into Shewy: <br />
Caleb, Tina, and a guest Louis ran a gel of the miniprep and colony parallel Phusion PCRs. All of the miniprep PCRs worked, but only a couple colony PCRs worked. We believe this is due to not having added enough cells to the colony PCR tube because some of the colonies were very tiny. <br />
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<b>Top: Positive control from colony, colony test 1, colony test 2, colony test 3, colony test 4, colony test 5, miniprep 5, Benchtop 1kb; Bottom: Benchtop 1kb, miniprep 1, miniprep 2, miniprep 3, miniprep 4, positive control miniprep:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c9/2012_8_25_Checking_for_nah_product_from_colony_vs_miniprep_PCRs.jpg/489px-2012_8_25_Checking_for_nah_product_from_colony_vs_miniprep_PCRs.jpg"><br />
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<h3>August 26th - 31st</h3><br />
Operation “get nah into Shewy continued this week.” We were not able to confirm through colony PCR that we have nah in WM3064. Team fluorescence was able to conjugate the SAL_mRFP constructs into Shewy. We continued site-directed mutagenesis of the nah operon but it has not been working like we expected. <br />
<div class="panel" style="background:white;margin-top: 20px;"><br />
<h6>Daily Details:</h6><br />
<br><b> August 26th, Sunday </b></br><br> Conjugating nah into Shewy: <br />
Caleb miniprepped two plasmids - one contained the nah operon preceded with a constitutive promoter and the other is to become a backbone containing the origin of transfer required for conjugation and a gene conveying chloramphenicol resistance. The purified plasmids were digested to extract the nah operon and to cut open the backbone - these digests were then run on a gel. Tina gel extracted the backbone then accidentally threw away the rest of the gel that contained the nah operon fragments. <br />
<br> Site-directed mutagenesis: Swati ran a mutagenic PCR of nah_p31c, then digested with DpnI to remove template DNA and column purified the resultant DNA. </br><br />
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<br><b> August 27th, Monday </b></br><br> Conjugating nah into Shewy: <br />
Caleb gel extracted the nah operon containing fragments from yesterday - despite having been in the garbage overnight, the gel extraction worked! Caleb and Tina began a 24 hour 16 degrees Celsius ligation using molar rations of nah to backbone of 3:1 and 1:1. <br />
Fluorescence tests: Claire started a JG700 + SAL2 culture and tried to do a PCR of SAL2_mRFP with sequencing primers to see if the band produced would correspond to the length of SAL2 plus the length of mRFP. Unfortunately, due to a mishap involving p10s vs. p2s and not checking what volume her pipette was set to, she ended up with a 70uL total volume and PCR failed. <br />
<br> Site-directed mutagenesis: Swati transformed the (hopefully) mutated DNA into DH5a. </br><br />
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<br><b> August 28th, Tuesday </b></br><br> Conjugating nah into Shewy: <br />
Caleb and Tina de-salted the 3:1 and 1:1 ligations, then transformed them into a DAP-requiring conjugation strain WM3064 via electroporation. The electroporated cells were plated on DAP + Cm plates. <br />
Fluorescence tests: JG700 once again takes a long time to grow, so it wasn’t until the evening that Claire miniprepped SAL2 from JG700. Tomorrow we will run another PCR and see if we have SAL2_mRFP, and SAL2 in JG700. <br />
<br> Site-directed mutagenesis: Swati's transformation did not work. After checking the PCR product and a PstI digest thereof on a gel, she discovered that the PCR was not working with high enough efficiency to produce a band on a gel. Upon consulting with Didi, our trusty post-doctoral adviser, she then restarted the mutagenic process with a PCR; this time, with a lower primer concentration and lower denaturation and annealing times. </br><br />
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<br><b> August 29th, Wednesday </b></br><br> Conjugating nah into Shewy: <br />
Caleb checked the transformation plates after ~14 hours in a 37 degrees Celsius incubator and noticed colonies, but the colonies were barely visible indicating very slow growth of the cells. <br />
Fluorescence tests: Set up a Taq PCR of SAL_mRFP, SAL2_mRFP and SAL2, with an extension time of 4.5 minutes and annealing temperature of 68degC. SAL2_mRFP has a band corresponding only to the length of SAL2, without mRFP, so we need to redo this ligation. However, the band from SAL_mRFP was the correct length (a little more than 4kb), so this ligation was successful and we can conjugate into Shewenella. Finally, the JG700 miniprep is the correct length for SAL2 (~3.8kb), so SAL2 has been successfully conjugated into Shewenella! <br />
<br> Site-directed mutagenesis: Swati digested her second mutagenic PCR with DpnI, column purified, and transformed into DH5a. </br><br />
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<br><b> August 30th, Thursday </b></br><br> Conjugating nah into Shewy: <br />
Tina checked the growth of yesterday's colonies and tried doing a colony PCR of each colony and a PCR of a positive control (same product, same primes, but in a different purified plasmid). The primers were designed to sit on the backbone and face into the oriT region and the site where we wanted to insert the nah operon. A PCR amplicon indicating successful ligation of the nah operon into the backbone would be approximately 10-11kb, while a failed ligation would yield a ~700 bp amplicon. Caleb later ran a DNA gel of the colony PCRs and nothing but smears showed up in every lane of the gel except the positive control which didn't even have a smear (Caleb was so disappointed that he didn't post the picture). Since the positive control didn't show up, we concluded the PCR failed. Caleb started overnight cultures of the remaining cells+ddH2O used for the colony PCR. <br />
<br> Site-directed mutagenesis: Alas, Swati's second attempt at mutagenesis was yet again fruitless. Concerned that the template may have been causing the problem, she performed a NotI digest and ran it on a gel alongside the latest mutagenic PCR. The template was fine; the PCR was, again, invisible. Swati grew extremely puzzled as to how her purified PCR product could have significant concentration upon quantification but yield no visible bands on a gel. She decided to try a third time, this time with longer denaturation and annealing times, but still the same concentration of primers.<br />
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<b>From left lane: Ladder, SAL2_mRFP (1), SAL2_mRFP (2), SAL2:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/f/fc/2012_08_30_gel.jpg/800px-2012_08_30_gel.jpg"><br />
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<br><b> August 31st, Friday </b></br><br> Conjugating nah into Shewy: Caleb noticed the overnight cultures didn't grow and concluded the cells must have died due to the cells being stored in an extremely hypotonic solution (ddH2O). He re-did colony PCRs of the colonies, a colony with a positive control, and a positive control of purified plasmid, but modified the colony PCR protocol. Instead of using the cells + ddH2O as template DNA, Caleb dipped the pipette tip into the colony, then dip and swirled it in the PCR rxn tube before dip and swirling into labeled microfuge tubes with 50 uL LB media. The microfuge tubes were stored in a 4 degrees Celsius fridge. A DNA gel of the colony PCRs showed that the ligation of nah into the backbone failed - as seen in the accompanying gel picture, our positive control worked (indicating the PCR conditions were fine), but the test colonies all only had 700 bp amplicons. <br />
<br />
Fluorescence tests: Claire found some old SAL2 and mRFP digested with PstI and SpeI already, so she decided to retry the ligation. She did a 40min ligation at room temp, and continued it overnight in the 4degC fridge, which the NEB rep said could increase efficience. However, since she forgot to dephosphorylate SAL2, we will never know if this room temp and overnight in the fridge combo would have been effective because it self ligated. <br />
<br> Site-directed mutagenesis: Swat ran a DpnI digestion of half of the previous day's PCR product, in an attempt to rule out the possibility that DpnI was functioning incorrectly. </br><br />
<br><br />
<b>From left lane: Ladder, positive control miniprepped plasmid, colony 1, colony 2, colony 3, colony 4, colony 5, colony 6, colony 7, colony 8, positive control colony:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/3/37/8_31_2012_WM3064_nah_EDIT.jpg/683px-8_31_2012_WM3064_nah_EDIT.jpg"><br />
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