Team:Penn/Notebook

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<title>Penn 2012 iGEM Wiki</title>
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<!----------------------------------------------------------------------------->
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<p style="text-align:center;color:white;">June 2012 Notebook</p>
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<div id="accordion-container">
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<h2 class="accordion-header">Week 1</h2>
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<p><b>June 6th</b></p>
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                                                <ul>
 +
                                                  <li>Set up some lab equipment</li>
 +
                                                  <li>Autoclaved for a while</li>
 +
                                                  <li>Organized biobrick stuff</li>
 +
                                                  <li>Called Vinoo about DNA planning</li>
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 +
                                                </ul> 
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<br>
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</script>
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<p><b>June 7th</b></p>
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                                                <ul>
 +
                                                  <li>Transformed Cph8, pLsr, and LuxS</li>
 +
                                                  <li>Placed order with Vinoo</li>
 +
<li>Developed idea using PGY/PCN system to activate a gene</li>
 +
                                                </ul>                   
 +
 +
 +
 +
</div>
 +
 +
<h2 class="accordion-header">Week 2</h2>
 +
 +
<div class="accordion-content">
 +
 +
<p><b>June 11th</b></p>
 +
                            &nbsp;<p>Wet Lab</p>
 +
                                                <ul>
 +
                                                  <li>PCR'd mCherry from NAS157</li>
 +
                                                  <li>Ran 1% Gel and purified product</li>
 +
                                                </ul>
 +
&nbsp;&nbsp;<p>Dry Lab</p>
 +
<ul>
 +
<li>Designed primers for LsR promoter</li>
 +
<li>Meeting with Dr. Sarkar</li>
 +
</ul>
 +
<br>
 +
<p><b>June 12th</b></p>
 +
                            &nbsp;<p>Wet Lab</p>
 +
                                                <ul>
 +
                                                  <li>Digested mCherry PCR product with BamHI and NotI</li>
 +
                                                  <li>Column purified mCherry and ligated into NAS152 backbone</li>
 +
<li>Transformed NAS152-mCherry into DH5alpha</li>
 +
<li>Poured 25 LB-Kan plates</li>
 +
                                                </ul>
 +
&nbsp;&nbsp;<p>Dry Lab</p>
 +
<ul>
 +
<li>Research more information about bacterial drug delivery system</li>
 +
<li>More research into biofilm project</li>
 +
</ul>
 +
<br>
 +
<p><b>June 14th</b></p>
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</head>
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&nbsp;&nbsp;<p>Dry Lab</p>
 +
<ul>
 +
<li>Met with Dr. Goulian, obtained pDawn and pDusk</li>
 +
<li>Identified inaK as a surface display gene we can use</li>
 +
</ul>
 +
</div>
 +
 +
<h2 class="accordion-header">Week 3</h2>
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<div class="accordion-content">
 +
 +
<p>June 18th</p>
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<body>
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&nbsp;&nbsp;<p>Wet Lab</p>
 +
<ul>
 +
<li>Miniprep pDawn and pDusk</li>
 +
<li>Test cut pDawn and pDusk with XmaI, analytical gel was correct</li>
 +
<li>Prep cut pDawn and pDusk with BamHI and NotI, gel purified</li>
 +
</ul>
 +
&nbsp;&nbsp;<p>Dry Lab</p>
 +
<ul>
 +
<li>Ordered and picked up PCR purification kit from cell center</li>
 +
<li>Additional orders through cell center</li>
 +
<li>Designed primers for one of Peter's components (forgot which)</li>
 +
</ul>
 +
<br>
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<div style="overflow:hidden;">
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<p>June 20</p>
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<div style="margin-bottom:0px; padding-bottom:0px;">
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<a href="https://2012.igem.org/Team:Penn" style="padding:0px; margin:0px;">
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<img src="https://static.igem.org/mediawiki/2012/e/e5/Bluelogo.jpg" /></a>
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</div>
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<ul class="nav">
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&nbsp;&nbsp;<p>Wet Lab</p>
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<li class="dropdown">
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<ul>
-
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<li>Picked 2 colonies of pDawn-mCherry, innoculated in 5 mL of LB and 50 ug/mL of Kan</li>
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    <a href="#">Drug Delivery</a>
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<li>PCR purified fragments (Peter), then ran gel?</li>
-
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</ul>
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<ul>
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-
        <li><a href='/Team:Penn/DrugDeliveryOverview'>Overview</a></li>
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-
<li><a href='/Team:Penn/DrugDeliverySystem'>System</a></li>
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-
<li><a href='/Team:Penn/DrugDeliveryResults'>Results</a></li>
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-
<li><a href='/Team:Penn/DrugDeliveryParts'>Parts</a></li>
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</ul>
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</li>
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-
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<li class="dropdown">
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-
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-
    <a href="#">Biofilms</a>
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<ul>
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-
              <li><a href='/Team:Penn/BiofilmsOverview'>Overview</a></li>
+
-
<li><a href='/Team:Penn/BioflmsSystem'>System</a></li>
+
-
<li><a href='/Team:Penn/BiofilmsResults'>Results</a></li>
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-
<li><a href='/Team:Penn/BiofilmsParts'>Parts</a></li>
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-
</ul>
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</li>
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-
<li class="dropdown">
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&nbsp;&nbsp;<p>Dry Lab</p>
-
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<ul>
-
<a href="#">Lab Work</a>
+
<li>Researched DARPin binding domains and linkers</li>
-
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<li>Finalized some biobrick orders</li>
-
<ul>
+
<li>Finalized synthesis order (minus linker)</li>
-
+
</ul>
-
                                <li><a href='/Team:Penn/Notebook'>Notebook</a></li>
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-
<li><a href='/Team:Penn/Safety'>Safety</a></li>
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-
<li><a href='/Team:Penn/Protocols'>Protocols</a></li>
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-
</ul>
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-
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        </li>
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<li class="dropdown">
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<a href="#">Human Practices</a>
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<ul>
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-
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<li><a href='/Team:Penn/Education'>Education</a></li>
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-
<li><a href='/Team:Penn/Regulations'>Regulations</a></li>
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-
<li><a href='/Team:Penn/Registry'>The Registry</a></li>
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</ul>
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</li>
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<li class="dropdown">
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-
<a href="#">About Us</a>
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<ul>
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-
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<li><a href='/Team:Penn/Team'>Team</a></li>
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<li><a href='/Team:Penn/Advisors'>Advisors</a></li>
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<li><a href='/Team:Penn/Sponsors'>Sponsors</a></li>
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</ul>
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</li>
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<div class="arrow"></div>
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</ul>
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<!----------------------------------------------------------------------------->
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<div id="accordion-container">
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</div>
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<h2 class="accordion-header">What is it?</h2>
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<h2 class="accordion-header">Week 4</h2>
<div class="accordion-content">
<div class="accordion-content">
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<p class="first-p">Vallenato is a super simple jQuery based accordion script. There are plenty of other great accordion scripts out there to choose from, so why use Vallenato? Well, it's easy to implement, looks good, is flexible, and of course...it's free. This page is an example of Vallenato in action!</p>
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<div id="tweet">
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<p><b>June 22</b></p>
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<a href="http://twitter.com/share" class="twitter-share-button" data-count="horizontal" data-via="switchroyale">Tweet</a><script type="text/javascript" src="http://platform.twitter.com/widgets.js"></script>
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</div>
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&nbsp;&nbsp;<p>Wet Lab</p>
-
+
<ul>
-
<div id="like">
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<li>Ashwin - Repeated miniprep on pDawn and did test cut</li>
-
<script src="http://connect.facebook.net/en_US/all.js#xfbml=1"></script><fb:like href="http://www.switchroyale.com/vallenato" layout="button_count" show_faces="false" width="450" font="verdana"></fb:like>
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<li>Peter  - Miniprepped pet26b and digested with BglII and EcorRI</li>
-
</div>
+
<li>Avin - Miniprepped pet26b, made glycerol stock and digested with BamH1 and Not1</li>
 +
</ul>
 +
&nbsp;&nbsp;<p>Dry Lab</p>
 +
<ul>
 +
<li>Avin - Finalized and sent in synthesis order (still awaiting order confirmation)</li>
 +
</ul>
 +
 
 +
<p><b>June 25</b></p>
 +
 
 +
&nbsp;&nbsp;<p>Wet Lab</p>
 +
<ul>
 +
<li>Ashwin/Avin - Column purification, ligation, and transformation of Pet26b-mCherry</li>
 +
<li>Peter -run gel of eGFP/plsr ligation</li>
 +
</ul>
 +
 
 +
&nbsp;&nbsp;<p>Dry Lab</p>
 +
<ul>
 +
<li>Avin - Sent in final gene synthesis order</li>
 +
<li>Mike - reviewed pDawn protocol, reviewed TetR sequences</li>
 +
<li>Peter- Order restriction enzymes from cell center</li>
 +
</ul>
 +
<br>
 +
 
 +
<p><b>June 26</b></p>
 +
&nbsp;&nbsp;<p>Wet Lab</p>
 +
<ul>
 +
<li>Avin - Picked colonies for Pet26b-mCherry and pJT106b</li>
 +
<li>Mike/Ashwin - Plated pJT122</li>
 +
</ul>
 +
&nbsp;&nbsp;<p>Dry Lab</p>
 +
<ul>
 +
<li>Everyone - Brainstormed human practices, Wiki design</li>
 +
</ul>
 +
<br>
 +
 
 +
 
 +
<p><b>June 27</b></p>
 +
 
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>Miniprepped pet26b-mCherry and pDawn-mCherry</li>
 +
<li>Test cut pet26b-mCherry with HindIII and ClaI (picture temporarily saved in MEAM folder of pqiao account), bands looked okay, chose C2 for remainder of experiments and made a glycerol stock, transformed both pet26b-mCherry and pDawn-mCherry into BL21(DE3)- Gold (10 ul cells, ~1ng DNA)
 +
Plated 200 ul and 20 ul</li>
 +
<li>Inoculated colonies for pJT122, pJT106b, PhyB (BBa), PIF3 (BBa), and Cph8 (BBa)</li>
 +
</ul>
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Started exploring possible wiki designs and coding</li>
 +
</ul>
 +
<br>
 +
<p><b>June 28</b></p>
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>Miniprepped pJT122, pJT106b, PhyB, PIF3, Cph8, made glycerol stocks</li>
 +
<li>Picked colonies for pET26b-mCherry and pDawn-mCherry, innoculated, grew up, then diluted
 +
Measured OD along the way</li>
 +
</ul>
 +
&nbsp; &nbsp; <p>Dry Lab:</p>
 +
<ul>
 +
<li>Set up light and dark incubators</li>
 +
</ul>
 +
 
 +
<p><b>June 29</b></p>
 +
 
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>Tested pDawn-mCherry and pET-mCherry, pDawn-mCherry expressed mCherry only after light induction</li>
 +
<li>Nanodropped pJT122, pJT106b, PhyB, PIF3, Cph8</li>
 +
<li>Performed digest of pET-26b, eGFP, and plsr</li>
 +
<li>Spread Biobrick shipment on Amp plates (lsrR, lsrK)</li>
 +
</ul>
 +
 
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Opened Bio-Rad shipments</li>
 +
</ul>
 +
<br>
 +
 
 +
 
 +
</div>
</div>
 +
</div>
 +
<p style="text-align:center; color:white;">July 2012 Notebook</p>
 +
<div id="accordion-container">
 +
<h2 class="accordion-header">Week 5</h2>
 +
 +
<div class="accordion-content">
 +
             
 +
<p><b>July 2</b></p>
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>Transformed ho1 and pcyA BioBricks</li>
 +
<li>Peter: ligations</li>
 +
</ul>
 +
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
 +
<li>Contacted labs for JT2 and pPL-PCB</li>
 +
<li>Worked on Human Practices</li>
 +
</ul>
 +
 +
<br>
 +
 +
 +
 +
<p><b>July 3</b></p>
 +
 +
&nbsp;&nbsp;<p>Wet Lab</p>
 +
<ul>
 +
<li>Nothing to be done for drug delivery</li>
 +
</ul>
 +
&nbsp;&nbsp;<p>Dry Lab</p>
 +
<ul>
 +
<li>Avin - digital schematic, helped with primers, read human practices bacterial therapy stuff</li>
 +
<li>Mike - talked to Dan, developed cloning strategy for getting Cph8 into the pDawn backbone, started primer design</li>
 +
</ul>
 +
</br>
 +
 +
 +
 +
<p><b>July 5</b></p>
 +
 +
&nbsp;&nbsp;<p>Wet Lab</p>
 +
<ul>
 +
<li>Picked colonies of ho1 and pcyA</li>
 +
<li>Inoculated pDawn-mCherry cells for timecourse</li>
 +
</ul>
 +
&nbsp;&nbsp;<p>Dry Lab</p>
 +
<ul>
 +
<li>worked on primer design/cloning strategy and talked to Dan</li>
 +
<li>worked on schematic and wiki</li>
 +
<li>worked on human practices</li>
 +
</ul>
 +
</br>
 +
 +
<p><b>July 6-7</b></p>
 +
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
 +
<li>Took pDawn-mCherry time course</li>
 +
<li>Sterilized incubator and TC hood</li>
 +
<li>Moved lab stuff to small room</li>
 +
<li>Redesigned primers for plsr & egfp</li>
 +
</ul>
 +
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>
 +
Contacted FDA contacts for human practices
 +
</li>
 +
</ul>
 +
<br>
-
<h2 class="accordion-header">Download</h2>
+
</div>
 +
 +
<h2 class="accordion-header">Week 6</h2>
<div class="accordion-content">
<div class="accordion-content">
-
<p class="first-p">Download the Vallenato script here:</p>
+
-
+
<p><b>July 9</b></p>
-
<p class="last-p"><a href="vallenato.zip">Vallenato</a></p>
+
 
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>Inoculated 50mL cultures of pDawn-mCherry</li>
 +
<li>Set up pH meter, picked up JT2</li>
 +
<li>Organized lab area </li>
 +
<li>Made bacterial streaks for biobricks</li>
 +
</ul>
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Ordered mammalian cell culture media</li>
 +
<li>Finalized wiki template</li>
 +
<li>Worked on cloning steps for cph8</li>
 +
</ul>
 +
<br>
 +
 
 +
 
 +
 
 +
<p><b>July 10</b></p>
 +
 
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>Did time course for pDawn-mCherry, but since it was from a glycerol stock, saw no growth → will let grow overnight, then dilute in the morning and start a new time course</li>
 +
<li>Picked colonies from transformed Biobricks and innoculated</li>
 +
<li>Resuspended ClyA and LuxS IDT DNA and transformed into DH5alpha</li>
 +
<li>TC/Sterile practice training</li>
 +
</ul>
 +
 
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Ordered primers for ClyA-RFP, Peters primers</li>
 +
<li>Worked on wiki</li>
 +
<li>Emailed Tabor for pJT106b plasmid map</li>
 +
</ul>
 +
<br>
 +
 
 +
 
 +
 
 +
<p><b>July 11</b></p>
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>
 +
Recorded BL21 pDawn-mCherry growth curve and calculated doubling time</li>
 +
<li>
 +
<ul> Miniprepped biobricks
 +
<li>
 +
BBa_K265008 - Ice Nucleation Protein NC
 +
</li>
 +
<li>
 +
BBa_K523013 - Plac + INP-EYFP
 +
</li>
 +
<li>
 +
BBa_K299810 - B0032 + Invasin
 +
</li>
 +
<li>
 +
BBa_K257010 - ClyA + RFP
 +
</li>
 +
</ul>
 +
</li>
 +
</ul>
 +
 
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Worked on biofilm project schematic</li>
 +
<li>More work on wiki</li>
 +
</ul>
 +
<br>
 +
 
 +
 
 +
 
 +
<p><b>July 13</b></p>
 +
 
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>Diluted to OD600=0.01 and take 0-8hr time points of pDawn-mCherry fluorescence time course</li>
 +
<li>Thawed out HEK293T cells</li>
 +
</ul>
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Worked on wiki/schematic</li>
 +
</ul>
 +
<br>
 +
 
 +
<p><b>July 17</b><p>
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
 
 +
<li>Re-ligation of pDawn with IDTClyA and ClyA-RFP, Transformation into DH5alpha/XO1blue</li>
 +
<li>Performed PCR of plsr & gfp w/new primers.</li>
 +
<li>Picked colonies of T7 and INPNC-DARPin</li>
 +
</ul>
 +
 
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Designed experiments for ClyA and INPNC-DARPin assays</li>
 +
</ul>
 +
<br>
 +
 
 +
 
</div>
</div>
-
<h2 class="accordion-header">Installation</h2>
+
<h2 class="accordion-header">Week 7</h2>
<div class="accordion-content">
<div class="accordion-content">
-
<p class="first-p">First, unzip the "vallenato.zip" file and place the "vallenato" folder in the same directory as the html file(s) that will be using the script. If placed in a different folder, you will need to update the paths below.</p>
+
-
+
<p><b>July 16</b></p>
-
<p>In the &lt;head&gt; section of your html you will need to link to jQuery, the Vallenato script and stylesheet. You can use the following code:</p>
+
&nbsp;&nbsp;<p>Wet Lab:</p>
-
+
<ul>
-
<div class="code">
+
<li>
-
<code>
+
Transformed INPNC-DARPin and T7 BioBrick
-
<p class="first-p">&lt;script src="http://code.jquery.com/jquery-latest.js" type="text/javascript"&gt;&lt;/script&gt;</p>
+
</li>
 +
</ul>
 +
 
 +
 
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>SAAST presentation</li>
 +
<li>Met with Lazzara lab to discuss DARPin experiments</li>
 +
</ul>
 +
<br>
 +
 
 +
<p><b>July 18</b></p>
 +
 
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>
 +
Re-inoculated T7 and INPNC-DARPin</li>
 +
<li>PCR ClyA-RFP, Gel purify PCR ClyA-RFP product</li>
 +
</ul>
 +
 
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Worked on DARPin assay experiment design</li>
 +
</ul>
 +
<br>
 +
 
 +
<p><b>July 19</b></p>
 +
 
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>Trypan Blue Assay</li>
 +
<li>Digested pDawn-mCherry with BamHI, NotI, pDawn-mCherry with BamHI,Digest pDawn-mCherry with NotI, Digested IDTsmart-ClyA with BamHI, NotI (Gel for Digests showed possible degeneration of NotI enzyme...) </li>
 +
<li>Ligated pDawn backbone (old) with 1) ClyA and 2) ClyA-RFP and Ligated pDawn backbone (new) with ClyA and ClyA-RFP, as well as pDawn old backbone with H20 and pDawn new backbone with H20 (ligation controls)</li>
 +
<li>Transformed all of above ligations into DH5alpha</li>
 +
</ul>
 +
<br>
 +
 
 +
 
 +
<p><b>July 23</b></p>
 +
 
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>Ashwin-Design test cuts for pDawn-Clya, pDawn-ClyA+RFP </li>
 +
<li>Ashwin-Digest pDawn-ClyA+pDawn-ClyA+RFP, Run on gel </li>
 +
<li>Nikita - Miniprep pDawn culture in Sarkar cold room</li>
 +
<li>Digest of pET-26b w/ BglII, EcoRI-Peter</li>
 +
<li>Digest of plsr product w/ BglII, PseI-Peter</li>
 +
<li>Digest egfp w/ SpeI, EcoRI-Peter</li>
 +
<li>Ligate & transform pET-26-plsr-GFP-Peter</li>
 +
<li>Digest INPNC-DARPin</li>
 +
<li>Avin - Try to rescue 293T cells after CO2 arrives if can’t be rescued, thaw out more 293T</li>
 +
<li>Transform pET-26b-luxS - Peter</li>
 +
<li>Avin - pick GAVPO </li>
 +
</ul>
 +
 
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Ordered AI-2 ASAP-peter</li>
 +
<li>Nikita - Researched whether ClyA is toxic to E. coli BL21, JT2, DH5 alpha, and whether it is secreted in these strains</li>
 +
<li>Read Daphne+Dr. Sarkar’s ligation paper </li>
 +
<li><ul>Avin - Buy:  
 +
<li>DH5 alpha</li>
 +
<li>20 kan plates, 10 amp plates</li>
 +
<li>Cell Counter</li>
 +
</ul>
 +
</li>
 +
<li>
 +
Write out competent cell production protocol-Peter</li>
 +
<li>Obtained BL21 transformation protocol from daphne/find it-Peter</li>
 +
<li>Avin - Logo design, human practices</li>
 +
<li>Ashwin - wiki</li>
 +
</ul>
 +
</br>
 +
 
 +
 
 +
 
 +
<p><b>July 24</b></p>
 +
 
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>Pick colonies of pDawn-ClyA and pDawn-ClyA+RFP (DH5alpha)</li>
 +
<li>Avin - Picked 2 colonies (light+dark) of pDawn-ClyA+RFP (BL21), set up pDawn culture experiment, check on HEK293T and passage into 6 well plates if confluent</li>
 +
<li>Avin - Ran Gel, no DNA</li>
 +
</ul>
 +
 
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Peter: order the promoter (or figure out an alternative method..)</li>
 +
<li>Ashwin: wiki</li>
 +
<li>Avin - Edit/Order primers for HA tag and His tag, design pDawn-ClyA experiments, human practices</li>
 +
<li>Nikita - Write human practices</li>
 +
</ul>
 +
</br>
-
<p>&lt;script src="vallenato/vallenato.js" type="text/javascript"&gt;&lt;/script&gt;</p>
 
-
<p class="last-p">&lt;link rel="stylesheet" href="vallenato/vallenato.css" type="text/css" media="screen"&gt;</p>
 
-
</code>
 
-
</div>
 
</div>
</div>
 +
<h2 class="accordion-header">Week 8</h2>
-
<h2 class="accordion-header">Mark Up</h2>
+
<div class="accordion-content">
 +
 
 +
<p><b>July 26</b></p>
 +
 
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>Avin - Finish ClyA-RFP time course, collect ClyA supernatant, measure fluorescence of ClyA-RFP triplicates, spin down + pictures if red, Miniprep of DARPin, possible ClyA assay</li>
 +
<li>Peter- ligation, transformation, plating of ligations, help Avin with ClyA cell lysis experiments </li>
 +
<li>Transform luxS6-8 into BL21 </li>
 +
<li> Nikita - Miniprep GAVPO and DARPin </li>
 +
</ul>
 +
 
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Avin - Call IDT at 9am, order blood agar plates, nissl stuff after we do background research</li>
 +
<li>Ashwin - wiki, research getting recombinant ClyA</li>
 +
<li>Nikita - Human practices, Nissle research, plan experiments</li>
 +
<li><ul>Peter
 +
<li>Complete FDA writeup</li>
 +
<li>Order ATCC Strains</li>
 +
<li>Order lss primers</li>
 +
<li>Order AI-2</li>
 +
<li>Order Crystal Violet Stain</li>
 +
<li>Plan ClyA experiment in JMOutline</li>
 +
</ul>
 +
</li>
 +
</ul>
 +
<br>
 +
 
 +
 
 +
 
 +
<p><b>July 30</b></p>
 +
 
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>Avin - Transform pBAD33 and pSB4A5, Inoculate BL21 Intein colonies</li>
 +
<li>Redo the test cuts for pDawn and pDawn-ClyA-RFP with XmaI and ClaI on a 0.7% gel</li>
 +
</ul>
 +
 
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Avin - Human Practices</li>
 +
<li><ul>Mike - Primers
 +
<li>pET26b-ClyA (IDT) keeping pelB with 6xHis C terminus - use NcoI on forward primer, figure out reverse</li>
 +
<li>pET26b-ClyA (IDT) removing pelB with 6xHis C terminus - use NdeI on forward primer, figure out reverse</li>
 +
<li>pet26b-ClyA+RFP keeping pelB with 6xHis C terminus - use NcoI on forward primer, figure out reverse</li>
 +
<li>pet26B-CLlyA+RFP removing pelB with 6x His C terminus - use NdeI on forward primer, figure out reverse</li>
 +
<li>fix frame shift in pDawn and include 6xHis N terminus - use NdeI on forward primer, figure out reverse</li>
 +
<li>ClyA </li>
 +
<li>mCherry</li>
 +
<li>ClyA-RFP</li>
 +
<li>Cph8 primers - figure out pJT106b primers</li>
 +
</ul>
 +
</li>
 +
</ul>
 +
<br>
 +
 
 +
<p><b>July 31</b></p>
 +
 
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>
 +
Test Cut pDawn-ClyA-RFP colony 4 w/ XmaI and Cla</li>
 +
<li>
 +
Transform the pDawn-ClyA-RFP colony 4 plasmid (assuming test cuts look good) into BL21 and Dh5alpha</li>
 +
<li>
 +
Check for growth of Intein-mCherry plasmid</li>
 +
<li><ul>
 +
Assuming growth:
 +
<li>miniprep DH5alpha culture</li>
 +
<li>induce BL21 culture with IPTG - see Jordan for protocol</li>
 +
<dilute in morning, when it gets to 0.8, induce with IPTG (what concentration) and then in a few hours (2-4), can spin down and see pellet</li>
 +
<li>If primers arrive → PCR (x2) for DARPin (check order status for IDT in the morning, call cell center if it says its been shipped)</li>
 +
<li>Pick colonies and innoculate pBAD33 (chloramphenicol) and pSB4A5 (ampicillin)</li>
 +
</ul>
 +
</li>
 +
</ul>
 +
 
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Mike - Order Primers<ul></li>
 +
<li>
 +
pET26b-ClyA (IDT) keeping pelB with 6xHis C terminus - use NcoI on forward primer, XhoI on reverse (no stop codon)</li>
 +
<li>
 +
pET26b-ClyA (IDT) removing pelB with 6xHis C terminus - use NdeI on forward primer, XhoI on reverse (no stop codon)
 +
</li>
 +
<li>
 +
fix frame shift in pET26b for ClyA-RFP (NdeI on forward, NotI on reverse, include stop) - can’t use NdeI because there is an NdeI cut site inside ClyA-RFP
 +
</li>
 +
<li>
 +
fix frame shift in pDawn and include 6xHis N terminus
 +
</li>
 +
<li>
 +
ClyA - NdeI on forward, BamHI on reverse
 +
</li>
 +
<li>
 +
mCherry - NdeI on forward, NotI on reverse
 +
</li>
 +
<li>
 +
ClyA-RFP - NdeI on forward, NotI on reverse - can’t use NdeI because there’s an NdeI cut site inside ClyA-RFP
 +
</li>
 +
<li>
 +
Cph8 primers - figure out pJT106b primers
 +
</li>
 +
</ul>
 +
</li>
 +
<li>
 +
Mike - Take out Biohazard for Sevile
 +
</li>
 +
<li>
 +
Ashwin - wiki
 +
</li>
 +
</ul>
 +
 
 +
<br>
 +
 
 +
 +
</div>
 +
</div>
 +
 
 +
<p style="text-align:center;color:white;">August 2012 Notebook</p>
 +
<div id="accordion-container">
 +
<h2 class="accordion-header">Week 9</h2>
<div class="accordion-content">
<div class="accordion-content">
-
<p class="first-p">Mark up is simple and straight forward, just use the following code as a template:</p>
 
-
 
-
</div>
 
 +
<p><b>August 1st</b></p>
 +
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<li>pET-26-plsr-gfp triple ligation ( (+) indicates positive control, (-) indicates negative control)</li>
 +
<li><ul>
 +
Gel Purification 8:30-12:00
 +
<li>
 +
1% gel 50mL +5uL SyberSafe</li>
 +
<li>
 +
pET-26b cut w/ EcoRI-HF (+)</li>
 +
<li>
 +
pET-26b cut w/ EcoRI-HF & BglII</li>
 +
<li>
 +
run against pET-26b uncut for reference
 +
</li></ul></li>
 +
<li><ul>
 +
Ligation (Start w/ 25ng vector) 12:00-3:00
 +
<li>
 +
Vector:GFP:plsr</li>
 +
<li>1:6:6</li>
 +
<li>1:4:4</li>
 +
<li>1:1:1</li>
 +
<li>pET-26b cut w/ EcoRI-HF (+)</li>
 +
<li>pET-26b cut w/ EcoRI-HF & BglII (-)</li>
 +
<li>INCUBATE @ RT 1hr</li>
 +
<li><ul>
 +
Transform Into DH5a Max Efficiency 3:00-6:00
 +
<li>Transform pET-26b (+)</li>
 +
<li>Transform H2O (-)</li>
 +
<li>Transform 1:6:6</li>
 +
<li>Transform 1:4:4</li>
 +
<li>Transform 1:1:1</li>
 +
</ul></li>
 +
<li>
 +
Total Plates Needed=8</li>
 +
<li>
 +
Check IDT primer order in the morning</li>
 +
<li>
 +
Avin - Pick colonies of pDawn-ClyA-RFP (DH5a and BL21) and inoculate into 5 mL LB culture
 +
</li>
 +
<li>
 +
Avin - Miniprep pSB4A5, pBAD33, Intein-mCherry (DH5a)
 +
Grow up BL21 Intein-mCherry and induce for fun?
 +
</li>
 +
</ul>
 +
</br>
 +
 +
<p><b>August 2</b></p>
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>
 +
pDawn-ClyA-RFP Time course - Avin </li>
 +
<li>
 +
growth to 0.8 and 1:1000 IPTG induction of 50mL BL21 Intein-mCherry - Avin </li>
 +
<li>
 +
Miniprep of DH5 alpha pDawn-ClyA-RFP - Ashwin </li>
 +
<li>
 +
PCR purify INPNC-DARPin-HA Tag - Mike </li>
 +
<li>
 +
Digest INPNC-DARPin-HA Tag and pET26b with EcoRI and NdeI
 +
</li>
 +
<li>
 +
Column purify INPNC-DARPin-HA Tag</li>
 +
<li>
 +
Gel purify pET26b </li>
 +
<li>
 +
Digest INPNC, INPNC-HA Tag, 6xHis-DARPin-Ha Tag with NdeI and BamHI </li>
 +
<li>Column purify INPNC, INPNC-HA Tag, 6xHis-DARPin-HA Tag</li>
 +
<li>
 +
Gel purify PET26b</li>
 +
<li>
 +
Gel purification of pET-26b digested with BglII and EcoRI-HF </li>
 +
<li>
 +
Plated S. Epidermis and E. Coli containing Lysostaphin</li>
 +
</ul>
 +
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Logo - Avin</li>
 +
</ul>
 +
<br>
 +
 +
<p><b>August 3</b></p>
 +
 +
&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>BL21 pDawn-ClyA-RFP time course, store blood agar plates in fridge - Avin</li>
 +
<li>BL21 Intein-mCherry Protein purification and Coomassie gel - Avin/Peter</li>
 +
<li>Ligate and transform pET26b-INPNC, pET26b-INPNC-HATag, pET26b-INPNC-DARPin-HATag, pET26b-6xHis-DARPin-HATag</li>
 +
<li>Saturday - pick colonies</li>
 +
<li>Sunday/Monday - Miniprep, test cuts, transformation</li>
 +
<li>Check if ClyA primers are shipped, if so pick up at cell center</li>
 +
<li>PCR ClyA with various primers</li>
 +
<li>Run on Gel, Gel Purify, Digest, Column Purify, Ligate, Transform</li>
 +
</ul>
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Pick up primers</li>
 +
<li>Meeting with Dr. Sarkar</li>
 +
</ul>
 +
<br>
 +
 +
 +
 +
 +
                        </div>
 +
 +
<h2 class="accordion-header">Week 10</h2>
 +
 +
<div class="accordion-content">
 +
 +
<p><b>August 13</b></p>
 +
<ul>
 +
<li>
 +
Started pDawn ClyA & pDawn ClyA-RFP cultures for blood agar experiment</li>
 +
<li>
 +
Meeting with Dr. Sarkar to discuss progress on both projects</li>
 +
</ul>
 +
<br>
 +
 +
 +
<p><b>August 14 </b></p>
 +
<ul>
 +
<li><ul>
 +
Reviewed sequencing results and transformed the following into BL21 and DH5 alpha:
 +
<li>
 +
pET26b-INPNC
 +
</li>
 +
<li>
 +
pET26b-INPNC-HA
 +
</li>
 +
<li>
 +
pET26b-6xHis-DARPin-HA
 +
</li>
 +
<li>
 +
pET26b-INPNC-DARPin-HA
 +
</li>
 +
<li>
 +
pET26b-ClyA-6xHis
 +
</li>
 +
<li>
 +
pET26b-PelB-ClyA-6xHis
 +
</li>
 +
<li>
 +
pDawn-ClyA-6xHis
 +
</li></ul></li>
 +
<li>
 +
Serial dilution and plating of pDawn ClyA & pDawn ClyA-RFP cultures for blood agar experiment</li>
 +
</ul>
 +
<br>
 +
 +
 +
 +
 +
 +
                        </div>
 +
 +
<h2 class="accordion-header">Week 11</h2>
 +
 +
<div class="accordion-content">
 +
 +
 +
<p><b>August 15</b></p>
 +
<ul>
 +
<li>Re-plated all constructs</li>
 +
<li>pDawn-ClyA and pDawn-ClyA-RFP demonstrated light-dependent hemolysis!</li>
 +
<li>BL21 washing titration experiment for flow cytometry experiment - obtained optimal starting OD600 of 0.05 to obtain ~1E6 cells after all washes and incubations</li>
 +
<li>Made Incubation Buffer for flow cytometry experiment</li>
 +
<li>Designed/ordered pBAD33-eGFP Primers</li>
 +
</ul>
 +
<br>
 +
 +
 +
 +
<p><b>August 16</b></p>
 +
<ul>
 +
<li>
 +
Picked colonies on all BL21 and DH5 constructs, including inducing and non-inducing conditions, set up flow cytometry stuff, booked flow core
 +
</li>
 +
<li>
 +
Started cultures of pDawn-ClyA, pDawn-ClyA-RFP, and pDawn-ClyA-mCherry for repeat blood agar experiment</li>
 +
<li>Set up light incubator properly</li>
 +
<li>Started protein purification cultures of pDawn-ClyA-6xHis, pET26b-ClyA-6xHis, and pET26b-PelB-ClyA-6xHis, grew to OD600=0.8, induced with IPTG or light</li>
 +
</ul>
 +
<br>
 +
 +
 +
<p><b>August 22 </b></p>
 +
<ul>
 +
<li>
 +
Image INPNC-DARPin-HA, INPNC-HA, DARPin-HA (both induced and not induced) on the confocal - Avin
 +
</li>
 +
<li>
 +
Figure out ClyA imaging system and maybe design a stencil (not a high priority)
 +
</li>
 +
<li>
 +
Pick up media and everything else from cell center - Mike
 +
</li>
 +
<li>
 +
Ligate ClyA into lactococcus? -- Talk to Daphne about vectors
 +
</li>
 +
<li>
 +
Order the cytotoxicity assay from Promega - Avin
 +
</li>
 +
<li>
 +
Ask Dr. Sarkar for cell center account - Mike
 +
</li>
 +
<li>
 +
Order pDawn sequencing primer - Avin
 +
</li>
 +
<li>
 +
Run GFP PCR product on Gel,
 +
Gel didn’t work → rerun PCR using gradient annealing temp
 +
Run PCR products on gel, check if works
 +
</li>
 +
<li>
 +
Look into biobrick format - Mike
 +
</li>
 +
<li>
 +
Make an in-depth plan for the incoming weeks, prioritizing what needs to be done
 +
Cloning! what is the final construct? Design and execute
 +
add RBS to pBAD33 primer
 +
</li>
 +
</ul>
 +
<br>
 +
 +
<p><b>Plan for August 23</b></p>
 +
<ul>
 +
<li>
 +
Add RBS/SD to pBAD33 forward primer and re-order it
 +
</li>
 +
<li>
 +
Send out sequencing for INPNC-DARPin-HA (and what else?)
 +
</li>
 +
<li>
 +
Look at old sequencing data just to make sure everything worked
 +
</li>
 +
<li>
 +
Pick up IPTG from the cell center and make stock solution/aliquots
 +
</li>
 +
<li>
 +
Using new IPTG, spread on blood agar plates (final dilution of 1 to 1000)
 +
</li>
 +
<li>
 +
Spread Kan on Blood Agar Plates
 +
</li>
 +
<li>
 +
Plate pET26b-ClyA-His (+/-), pET26b-pelB-ClyA-His (+/-), and pDawn-ClyA(FS) (+/-) on Blood Agar + Kan plates
 +
</li>
 +
<li>
 +
</ul>
 +
<br>
 +
 +
 +
<p><b>August 24</b></p>
 +
<ul>
 +
<li>
 +
If colonies were picked, miniprep, then test cut, then run on diagnostic gel → transform into BL21</li>
 +
<li>
 +
If colonies were not picked, then pick colonies</li>
 +
<li>
 +
Analyze sequencing results</li>
 +
<li>
 +
Plan out INPNC-DARPin-HA experiments
 +
</li>
 +
<li>
 +
Figure out cloning for 1 plasmid system
 +
</li>
 +
</ul>
 +
<br>
 +
 +
 +
 +
  </div>
 +
 +
<h2 class="accordion-header">Week 12</h2>
 +
 +
<div class="accordion-content">
 +
 +
<p><b>Goals in order of priority:</b></p>
 +
<ul>
 +
<li>
 +
Show that INPNC-DARPin-HA binds to HER2 in vitro
 +
</li>
 +
<li>
 +
Show that purified 6xHis-DARPin-HA binds to Her2 - Monday?
 +
</li>
 +
<li>
 +
Show that INPNC-DARPin-HA is displayed on the surface
 +
</li>
 +
<li>
 +
Show INPNC-mCherry is displayed on surface (confocal?)
 +
</li>
 +
<li>
 +
Construct ClyA and ClyA-His BioBricks
 +
</li>
 +
<li>
 +
Construct INPNC-mCherry Biobrick
 +
</li>
 +
<li>
 +
Construct Surface Display BioBrick (need INPNC-mCherry to work)
 +
</li>
 +
<li>
 +
Transform ClyA into Lactococcus and plate onto blood agar for Human Practices-
 +
</li>
 +
<li>
 +
Stencil experiment showing spatial control of pDawn-ClyA (could be done next week, only takes a little time and
 +
artistic ability)
 +
</li>
 +
<li>
 +
Show that cph8 works with a reporter
 +
</li>
 +
<li>
 +
Show that cph8-ClyA works
 +
</li>
 +
</ul>
 +
 +
<p><b>Experiments in order of priority:</b></p>
 +
<ul>
 +
<li>
 +
Construction of pBAD33-eGFP (Mike)
 +
</li>
 +
<li>
 +
Order primers for ClyA-His and ClyA to clone them into BioBrick backbone
 +
</li>
 +
<li>
 +
Order primers for INPNC-mCherry
 +
</li>
 +
<li>
 +
Order primers for cph8-reporter, cph8-clyA-his
 +
</li>
 +
<li>
 +
SKBR3 imaging with pBAD33-eGFP/pET26b-INPNC-DARPin-HA bacteria on confocal
 +
</li>
 +
<li>
 +
INPNC-DARPin immunos, 0.2mM IPTG, overnight induction
 +
</li>
 +
<li>
 +
INPNC-DARPin bacterial flow cytometry on FACS machine (can use same bacteria as immuno)
 +
</li>
 +
<li>
 +
Create stable cell line of SKBR3-mCherry (Jordan)
 +
</li>
 +
<li>
 +
Obtain Lactococcus expression vector, order primers to clone in ClyA-His
 +
</li>
 +
<li>
 +
Design surface display biobrick
 +
</li>
 +
<li>
 +
Cytotoxicity experiment on SKBR3</li>
 +
<li>
 +
Protein purification on His-DARPin-HA (Peter)
 +
</li>
 +
<li>
 +
Flow cytometry of SKBR3 cells incubated with his-DARPin-HA (someone/Najaf)
 +
</li>
 +
<li>
 +
Construct cph8-ClyA plasmid
 +
</li>
 +
<li>
 +
cph8-reporter timecourse
 +
</li>
 +
<li>
 +
cph8-ClyA blood agar
 +
</li>
 +
</ul>
 +
<br>
 +
 +
<p><b>August 27</b></p>
 +
<ul>
 +
<li>
 +
PCR eGFP out of PHAT (annealing = 65), run on 1% gel, gel purify
 +
</li>
 +
<li>
 +
Digest eGFP and pBAD33 with PstI and XmaI, column purify
 +
</li>
 +
<li>
 +
Ligate at RT for 1 hour, transform into DH5a
 +
depending on time, otherwise ligate at 16C/4C overnight and transform the next day
 +
</li>
 +
<li>
 +
Order Primers for (in order of priority)
 +
</li>
 +
<li>
 +
ClyA and ClyA-His Biobricks
 +
</li>
 +
<li>
 +
INPNC-mCherry construct
 +
</li>
 +
<li>
 +
ClyA into lactococcus plasmid
 +
</li>
 +
<li>
 +
Cph8-reporter plasmid
 +
</li>
 +
<li>
 +
Cph8-ClyA-His plasmid
 +
</li>
 +
<li>
 +
INPNC surface display vector (provided it works)
 +
</li>
 +
<li>
 +
Start cell culture of SK-BR-3 cells?
 +
</li>
 +
<li>
 +
Get protocols
 +
</li>
 +
</ul>
 +
<br>
 +
 +
<p><b>August 28</b></p>
 +
<ul>
 +
<li>
 +
start cultures for purification of His-DARPin-HA
 +
</li>
 +
<li>
 +
HA affinity purification of INPNC-DARPin-HA?
 +
</li>
 +
<li>
 +
Print and hand in safety form to EHRS
 +
</li>
 +
<li>
 +
Pick up SK-BR-3 plate from Cal, learn protocols
 +
</li>
 +
<li>
 +
Add FBS to our McCoy’s media
 +
</li>
 +
<li>
 +
Design and order primers to put ClyA into lactococcus expression vector
 +
</li>
 +
<li>
 +
Set up preliminary stencil experiment, send avin plate base diameter (approximate 86mm)
 +
</li>
 +
<li>
 +
Design INPNC-mCherry-DARPin primers
 +
</li>
 +
<li>
 +
pBAD33-eGFP
 +
</li>
 +
<li>
 +
Run Gradient PCR  products (which ran overnight) on gel, gel purify the best band
 +
</li>
 +
<li>
 +
Digest eGFP and pBAD33 with PstI and XmaI, column purify/gel purify
 +
</li>
 +
<li>
 +
Ligate at RT for 1 hour, transform into DH5a
 +
depending on time, otherwise ligate at 16C/4C overnight and transform the next day
 +
</li>
 +
<li>
 +
Start DH5 alpha cultures of INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin-HA</li><li> Also start cultures of pET-ClyA-His, pET-PelB-ClyA-His and pDawn-ClyA-His for minipreps
 +
</li>
 +
</ul>
 +
<br>
 +
 +
<p><b>August 29</b></p>
 +
<ul>
 +
<li>
 +
Induce His-DARPin-HA culture (100 microliters of 1 M IPTG)</li>
 +
<li>
 +
Monitor SK-BR-3 </li>
 +
<li>
 +
Primers for ClyA into lactococcus</li>
 +
<li>
 +
Primers for INPNC-mCherry-DARPin </li>
 +
<li>
 +
Pick colonies for pBAD33-eGFP if the RT ligation and transformation worked - Peter/Ashwin</li>
 +
<li>
 +
If RT ligation and transformation looks bad, transform the 4C and 16C ligations - Mike</li>
 +
<li>
 +
When primers arrive, PCR out mCherry from JMIL intein plasmid, PCR out INPNC, then assembly PCR for INPNC-mCherry</li>
 +
</ul>
 +
<br>
 +
 +
 +
<p><b>August 30</b></p>
 +
<ul>
 +
<li>
 +
Protein purification of His-DARPin-HA
 +
</li>
 +
<li>
 +
PCR purify INPNC-mCherry, run a few ul on a diagnostic gel → results call for gel purifiation → Gel purify INPNC-mCherry → yield was bad, redo PCR  → gel purify
 +
</li>
 +
<li>
 +
Digest INPNC-mCherry and pET26b with NdeI and HindIII, ligate at RT for 1 hr (and maybe some at 4C or 16C overnight), and transform into pET26b
 +
</li>
 +
<li>
 +
Start DH5a cultures for INPNC, INPNC-HA, His-DARPin-HA, pET-ClyA-His, pET-PelB-ClyA-His and pDawn-ClyA-His for minipreps
 +
</li>
 +
<li>
 +
Repick colonies of pBAD33-eGFP from 16C or 4C plate
 +
</li>
 +
</ul>
 +
<br>
 +
                   
 +
  </div>
 +
 +
</div>
 +
 +
 +
 +
<p style="text-align:center;color:white;">September 2012 Notebook</p>
 +
<div id="accordion-container">
 +
<h2 class="accordion-header">Week 13</h2>
 +
<div class="accordion-content">
 +
 +
                        </div>
 +
 +
<h2 class="accordion-header">Week 14</h2>
 +
 +
<div class="accordion-content">
 +
 +
<p><b>September 10</b></p>
 +
<ul>
 +
<li>
 +
Mike - pick INPNC-mCherry colony (transformed Sample #2) and split into + and - cultures
 +
</li>
 +
<li>Start BL21 cultures of INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin-HA, His-Intein-mCherry
 +
</li>
 +
<li>
 +
Peter - Protein purification of His-DARPin-HA, ClyA-His
 +
</li>
 +
<li>
 +
Avin - Dilute (9am) & Induce (?) His-DARPin-HA, INPNC-HA, INPNC-DARPin-HA at 0.5
 +
</li>
 +
<li>
 +
Ashwin - Wiki work
 +
</li>
 +
</ul>
 +
<br>
 +
 +
<p><b>September 11</b></p>
 +
<ul>
 +
<li>
 +
Mike - Make glycerol stock of INPNC-mCherry, Dilute and induce INPNC-mCherry, INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin HA, His-Intein-mCherry
 +
</li>
 +
<li>
 +
Peter - Make electro/chemically competent cells, transform
 +
</li>
 +
<li>
 +
Ashwin - Pick up S. typhimurium from Mark Goulian
 +
</li>
 +
</ul>
 +
<br>
 +
 +
<p><b>September 12</b></p>
 +
<ul>
 +
<li>
 +
Peter - Lysis and spin down (for INPNC-mCherry, aliquot  1mL of both (+) and (-) for Avin
 +
</li>
 +
<li>Peter- spin down and check if red, then lyse) , put in 4C, check Nissle eGFP colonies
 +
</li>
 +
<li>
 +
Avin - Immuno experiment on His-DARPin-HA, INPNC-HA, INPNC-DARPin-HA, Take INPNC-mCherry, His-Intein-mCherry, fix and mount onto scope slides
 +
</li>
 +
</ul>
 +
</br>
 +
 +
<p><b>September 13</b></p>
 +
<ul>
 +
<li>
 +
Mike - Bradford assay, Run protein gel</li>
 +
<li>Peter - </li>
 +
<li>
 +
Avin - Observe BL21 transformatons under confocal
 +
</li>
 +
<li>
 +
Ashwin - colony PCR on S. typhimurium for MisL
 +
</li>
 +
</ul>
 +
<br>
 +
 +
<p><b>September 14</b></p>
 +
<ul>
 +
<li>
 +
Mike -
 +
</li>
 +
<li>
 +
Peter -
 +
</li>
 +
<li>
 +
Avin - core confocal?
 +
</li>
 +
<li>
 +
Ashwin -
 +
</li>
 +
</ul>
 +
 +
&nbsp;&nbsp;<p>Other<p>
 +
<ul>
 +
<li>Order scfv plasmid, scfv primers</li>
 +
<li>
 +
Order MisL Primers, pick up misL, PCR on S. typhimurium
 +
</li>
 +
<li>
 +
Chemical/Electrocompetent Nissle
 +
</li>
 +
<li>
 +
Make LB, Glycerol
 +
</li>
 +
<li>
 +
Wiki
 +
</li>
 +
</ul>
 +
</br>
 +
                        </div>
 +
 +
<h2 class="accordion-header">Week 15</h2>
 +
 +
<div class="accordion-content">
 +
 +
                        </div>
 +
 +
<h2 class="accordion-header">Week 16</h2>
 +
 +
<div class="accordion-content">
 +
 +
                        </div>
 +
 +
 +
</div>

Latest revision as of 05:52, 21 October 2012

Penn 2012 iGEM Wiki

Image Map

June 2012 Notebook

Week 1

June 6th

  • Set up some lab equipment
  • Autoclaved for a while
  • Organized biobrick stuff
  • Called Vinoo about DNA planning

June 7th

  • Transformed Cph8, pLsr, and LuxS
  • Placed order with Vinoo
  • Developed idea using PGY/PCN system to activate a gene

Week 2

June 11th

 

Wet Lab

  • PCR'd mCherry from NAS157
  • Ran 1% Gel and purified product
  

Dry Lab

  • Designed primers for LsR promoter
  • Meeting with Dr. Sarkar

June 12th

 

Wet Lab

  • Digested mCherry PCR product with BamHI and NotI
  • Column purified mCherry and ligated into NAS152 backbone
  • Transformed NAS152-mCherry into DH5alpha
  • Poured 25 LB-Kan plates
  

Dry Lab

  • Research more information about bacterial drug delivery system
  • More research into biofilm project

June 14th

  

Dry Lab

  • Met with Dr. Goulian, obtained pDawn and pDusk
  • Identified inaK as a surface display gene we can use

Week 3

June 18th

  

Wet Lab

  • Miniprep pDawn and pDusk
  • Test cut pDawn and pDusk with XmaI, analytical gel was correct
  • Prep cut pDawn and pDusk with BamHI and NotI, gel purified
  

Dry Lab

  • Ordered and picked up PCR purification kit from cell center
  • Additional orders through cell center
  • Designed primers for one of Peter's components (forgot which)

June 20

  

Wet Lab

  • Picked 2 colonies of pDawn-mCherry, innoculated in 5 mL of LB and 50 ug/mL of Kan
  • PCR purified fragments (Peter), then ran gel?
  

Dry Lab

  • Researched DARPin binding domains and linkers
  • Finalized some biobrick orders
  • Finalized synthesis order (minus linker)

Week 4

June 22

  

Wet Lab

  • Ashwin - Repeated miniprep on pDawn and did test cut
  • Peter - Miniprepped pet26b and digested with BglII and EcorRI
  • Avin - Miniprepped pet26b, made glycerol stock and digested with BamH1 and Not1
  

Dry Lab

  • Avin - Finalized and sent in synthesis order (still awaiting order confirmation)

June 25

  

Wet Lab

  • Ashwin/Avin - Column purification, ligation, and transformation of Pet26b-mCherry
  • Peter -run gel of eGFP/plsr ligation
  

Dry Lab

  • Avin - Sent in final gene synthesis order
  • Mike - reviewed pDawn protocol, reviewed TetR sequences
  • Peter- Order restriction enzymes from cell center

June 26

  

Wet Lab

  • Avin - Picked colonies for Pet26b-mCherry and pJT106b
  • Mike/Ashwin - Plated pJT122
  

Dry Lab

  • Everyone - Brainstormed human practices, Wiki design

June 27

  

Wet Lab:

  • Miniprepped pet26b-mCherry and pDawn-mCherry
  • Test cut pet26b-mCherry with HindIII and ClaI (picture temporarily saved in MEAM folder of pqiao account), bands looked okay, chose C2 for remainder of experiments and made a glycerol stock, transformed both pet26b-mCherry and pDawn-mCherry into BL21(DE3)- Gold (10 ul cells, ~1ng DNA) Plated 200 ul and 20 ul
  • Inoculated colonies for pJT122, pJT106b, PhyB (BBa), PIF3 (BBa), and Cph8 (BBa)
  

Dry Lab:

  • Started exploring possible wiki designs and coding

June 28

  

Wet Lab:

  • Miniprepped pJT122, pJT106b, PhyB, PIF3, Cph8, made glycerol stocks
  • Picked colonies for pET26b-mCherry and pDawn-mCherry, innoculated, grew up, then diluted Measured OD along the way
   

Dry Lab:

  • Set up light and dark incubators

June 29

  

Wet Lab:

  • Tested pDawn-mCherry and pET-mCherry, pDawn-mCherry expressed mCherry only after light induction
  • Nanodropped pJT122, pJT106b, PhyB, PIF3, Cph8
  • Performed digest of pET-26b, eGFP, and plsr
  • Spread Biobrick shipment on Amp plates (lsrR, lsrK)
  

Dry Lab:

  • Opened Bio-Rad shipments

July 2012 Notebook

Week 5

July 2

  

Wet Lab:

  • Transformed ho1 and pcyA BioBricks
  • Peter: ligations
  

Dry Lab:

  • Contacted labs for JT2 and pPL-PCB
  • Worked on Human Practices

July 3

  

Wet Lab

  • Nothing to be done for drug delivery
  

Dry Lab

  • Avin - digital schematic, helped with primers, read human practices bacterial therapy stuff
  • Mike - talked to Dan, developed cloning strategy for getting Cph8 into the pDawn backbone, started primer design

July 5

  

Wet Lab

  • Picked colonies of ho1 and pcyA
  • Inoculated pDawn-mCherry cells for timecourse
  

Dry Lab

  • worked on primer design/cloning strategy and talked to Dan
  • worked on schematic and wiki
  • worked on human practices

July 6-7

  

Wet Lab:

  • Took pDawn-mCherry time course
  • Sterilized incubator and TC hood
  • Moved lab stuff to small room
  • Redesigned primers for plsr & egfp
  

Dry Lab:

  • Contacted FDA contacts for human practices

Week 6

July 9

  

Wet Lab:

  • Inoculated 50mL cultures of pDawn-mCherry
  • Set up pH meter, picked up JT2
  • Organized lab area
  • Made bacterial streaks for biobricks
  

Dry Lab:

  • Ordered mammalian cell culture media
  • Finalized wiki template
  • Worked on cloning steps for cph8

July 10

  

Wet Lab:

  • Did time course for pDawn-mCherry, but since it was from a glycerol stock, saw no growth → will let grow overnight, then dilute in the morning and start a new time course
  • Picked colonies from transformed Biobricks and innoculated
  • Resuspended ClyA and LuxS IDT DNA and transformed into DH5alpha
  • TC/Sterile practice training
  

Dry Lab:

  • Ordered primers for ClyA-RFP, Peters primers
  • Worked on wiki
  • Emailed Tabor for pJT106b plasmid map

July 11

  

Wet Lab:

  • Recorded BL21 pDawn-mCherry growth curve and calculated doubling time
    • Miniprepped biobricks
    • BBa_K265008 - Ice Nucleation Protein NC
    • BBa_K523013 - Plac + INP-EYFP
    • BBa_K299810 - B0032 + Invasin
    • BBa_K257010 - ClyA + RFP
  

Dry Lab:

  • Worked on biofilm project schematic
  • More work on wiki

July 13

  

Wet Lab:

  • Diluted to OD600=0.01 and take 0-8hr time points of pDawn-mCherry fluorescence time course
  • Thawed out HEK293T cells
  

Dry Lab:

  • Worked on wiki/schematic

July 17

  

Wet Lab:

  • Re-ligation of pDawn with IDTClyA and ClyA-RFP, Transformation into DH5alpha/XO1blue
  • Performed PCR of plsr & gfp w/new primers.
  • Picked colonies of T7 and INPNC-DARPin
  

Dry Lab:

  • Designed experiments for ClyA and INPNC-DARPin assays

Week 7

July 16

  

Wet Lab:

  • Transformed INPNC-DARPin and T7 BioBrick
  

Dry Lab:

  • SAAST presentation
  • Met with Lazzara lab to discuss DARPin experiments

July 18

  

Wet Lab:

  • Re-inoculated T7 and INPNC-DARPin
  • PCR ClyA-RFP, Gel purify PCR ClyA-RFP product
  

Dry Lab:

  • Worked on DARPin assay experiment design

July 19

  

Wet Lab:

  • Trypan Blue Assay
  • Digested pDawn-mCherry with BamHI, NotI, pDawn-mCherry with BamHI,Digest pDawn-mCherry with NotI, Digested IDTsmart-ClyA with BamHI, NotI (Gel for Digests showed possible degeneration of NotI enzyme...)
  • Ligated pDawn backbone (old) with 1) ClyA and 2) ClyA-RFP and Ligated pDawn backbone (new) with ClyA and ClyA-RFP, as well as pDawn old backbone with H20 and pDawn new backbone with H20 (ligation controls)
  • Transformed all of above ligations into DH5alpha

July 23

  

Wet Lab:

  • Ashwin-Design test cuts for pDawn-Clya, pDawn-ClyA+RFP
  • Ashwin-Digest pDawn-ClyA+pDawn-ClyA+RFP, Run on gel
  • Nikita - Miniprep pDawn culture in Sarkar cold room
  • Digest of pET-26b w/ BglII, EcoRI-Peter
  • Digest of plsr product w/ BglII, PseI-Peter
  • Digest egfp w/ SpeI, EcoRI-Peter
  • Ligate & transform pET-26-plsr-GFP-Peter
  • Digest INPNC-DARPin
  • Avin - Try to rescue 293T cells after CO2 arrives if can’t be rescued, thaw out more 293T
  • Transform pET-26b-luxS - Peter
  • Avin - pick GAVPO
  

Dry Lab:

  • Ordered AI-2 ASAP-peter
  • Nikita - Researched whether ClyA is toxic to E. coli BL21, JT2, DH5 alpha, and whether it is secreted in these strains
  • Read Daphne+Dr. Sarkar’s ligation paper
    • Avin - Buy:
    • DH5 alpha
    • 20 kan plates, 10 amp plates
    • Cell Counter
  • Write out competent cell production protocol-Peter
  • Obtained BL21 transformation protocol from daphne/find it-Peter
  • Avin - Logo design, human practices
  • Ashwin - wiki

July 24

  

Wet Lab:

  • Pick colonies of pDawn-ClyA and pDawn-ClyA+RFP (DH5alpha)
  • Avin - Picked 2 colonies (light+dark) of pDawn-ClyA+RFP (BL21), set up pDawn culture experiment, check on HEK293T and passage into 6 well plates if confluent
  • Avin - Ran Gel, no DNA
  

Dry Lab:

  • Peter: order the promoter (or figure out an alternative method..)
  • Ashwin: wiki
  • Avin - Edit/Order primers for HA tag and His tag, design pDawn-ClyA experiments, human practices
  • Nikita - Write human practices

Week 8

July 26

  

Wet Lab:

  • Avin - Finish ClyA-RFP time course, collect ClyA supernatant, measure fluorescence of ClyA-RFP triplicates, spin down + pictures if red, Miniprep of DARPin, possible ClyA assay
  • Peter- ligation, transformation, plating of ligations, help Avin with ClyA cell lysis experiments
  • Transform luxS6-8 into BL21
  • Nikita - Miniprep GAVPO and DARPin
  

Dry Lab:

  • Avin - Call IDT at 9am, order blood agar plates, nissl stuff after we do background research
  • Ashwin - wiki, research getting recombinant ClyA
  • Nikita - Human practices, Nissle research, plan experiments
    • Peter
    • Complete FDA writeup
    • Order ATCC Strains
    • Order lss primers
    • Order AI-2
    • Order Crystal Violet Stain
    • Plan ClyA experiment in JMOutline

July 30

  

Wet Lab:

  • Avin - Transform pBAD33 and pSB4A5, Inoculate BL21 Intein colonies
  • Redo the test cuts for pDawn and pDawn-ClyA-RFP with XmaI and ClaI on a 0.7% gel
  

Dry Lab:

  • Avin - Human Practices
    • Mike - Primers
    • pET26b-ClyA (IDT) keeping pelB with 6xHis C terminus - use NcoI on forward primer, figure out reverse
    • pET26b-ClyA (IDT) removing pelB with 6xHis C terminus - use NdeI on forward primer, figure out reverse
    • pet26b-ClyA+RFP keeping pelB with 6xHis C terminus - use NcoI on forward primer, figure out reverse
    • pet26B-CLlyA+RFP removing pelB with 6x His C terminus - use NdeI on forward primer, figure out reverse
    • fix frame shift in pDawn and include 6xHis N terminus - use NdeI on forward primer, figure out reverse
    • ClyA
    • mCherry
    • ClyA-RFP
    • Cph8 primers - figure out pJT106b primers

July 31

  

Wet Lab:

  • Test Cut pDawn-ClyA-RFP colony 4 w/ XmaI and Cla
  • Transform the pDawn-ClyA-RFP colony 4 plasmid (assuming test cuts look good) into BL21 and Dh5alpha
  • Check for growth of Intein-mCherry plasmid
    • Assuming growth:
    • miniprep DH5alpha culture
    • induce BL21 culture with IPTG - see Jordan for protocol
    • If primers arrive → PCR (x2) for DARPin (check order status for IDT in the morning, call cell center if it says its been shipped)
    • Pick colonies and innoculate pBAD33 (chloramphenicol) and pSB4A5 (ampicillin)
  

Dry Lab:

  • Mike - Order Primers
    • pET26b-ClyA (IDT) keeping pelB with 6xHis C terminus - use NcoI on forward primer, XhoI on reverse (no stop codon)
    • pET26b-ClyA (IDT) removing pelB with 6xHis C terminus - use NdeI on forward primer, XhoI on reverse (no stop codon)
    • fix frame shift in pET26b for ClyA-RFP (NdeI on forward, NotI on reverse, include stop) - can’t use NdeI because there is an NdeI cut site inside ClyA-RFP
    • fix frame shift in pDawn and include 6xHis N terminus
    • ClyA - NdeI on forward, BamHI on reverse
    • mCherry - NdeI on forward, NotI on reverse
    • ClyA-RFP - NdeI on forward, NotI on reverse - can’t use NdeI because there’s an NdeI cut site inside ClyA-RFP
    • Cph8 primers - figure out pJT106b primers
  • Mike - Take out Biohazard for Sevile
  • Ashwin - wiki

August 2012 Notebook

Week 9

August 1st

  

Wet Lab:

  • pET-26-plsr-gfp triple ligation ( (+) indicates positive control, (-) indicates negative control)
    • Gel Purification 8:30-12:00
    • 1% gel 50mL +5uL SyberSafe
    • pET-26b cut w/ EcoRI-HF (+)
    • pET-26b cut w/ EcoRI-HF & BglII
    • run against pET-26b uncut for reference
    • Ligation (Start w/ 25ng vector) 12:00-3:00
    • Vector:GFP:plsr
    • 1:6:6
    • 1:4:4
    • 1:1:1
    • pET-26b cut w/ EcoRI-HF (+)
    • pET-26b cut w/ EcoRI-HF & BglII (-)
    • INCUBATE @ RT 1hr
      • Transform Into DH5a Max Efficiency 3:00-6:00
      • Transform pET-26b (+)
      • Transform H2O (-)
      • Transform 1:6:6
      • Transform 1:4:4
      • Transform 1:1:1
    • Total Plates Needed=8
    • Check IDT primer order in the morning
    • Avin - Pick colonies of pDawn-ClyA-RFP (DH5a and BL21) and inoculate into 5 mL LB culture
    • Avin - Miniprep pSB4A5, pBAD33, Intein-mCherry (DH5a) Grow up BL21 Intein-mCherry and induce for fun?

    August 2

      

    Wet Lab:

    • pDawn-ClyA-RFP Time course - Avin
    • growth to 0.8 and 1:1000 IPTG induction of 50mL BL21 Intein-mCherry - Avin
    • Miniprep of DH5 alpha pDawn-ClyA-RFP - Ashwin
    • PCR purify INPNC-DARPin-HA Tag - Mike
    • Digest INPNC-DARPin-HA Tag and pET26b with EcoRI and NdeI
    • Column purify INPNC-DARPin-HA Tag
    • Gel purify pET26b
    • Digest INPNC, INPNC-HA Tag, 6xHis-DARPin-Ha Tag with NdeI and BamHI
    • Column purify INPNC, INPNC-HA Tag, 6xHis-DARPin-HA Tag
    • Gel purify PET26b
    • Gel purification of pET-26b digested with BglII and EcoRI-HF
    • Plated S. Epidermis and E. Coli containing Lysostaphin
      

    Dry Lab:

    • Logo - Avin

    August 3

      

    Wet Lab:

    • BL21 pDawn-ClyA-RFP time course, store blood agar plates in fridge - Avin
    • BL21 Intein-mCherry Protein purification and Coomassie gel - Avin/Peter
    • Ligate and transform pET26b-INPNC, pET26b-INPNC-HATag, pET26b-INPNC-DARPin-HATag, pET26b-6xHis-DARPin-HATag
    • Saturday - pick colonies
    • Sunday/Monday - Miniprep, test cuts, transformation
    • Check if ClyA primers are shipped, if so pick up at cell center
    • PCR ClyA with various primers
    • Run on Gel, Gel Purify, Digest, Column Purify, Ligate, Transform
      

    Dry Lab:

    • Pick up primers
    • Meeting with Dr. Sarkar

    • Week 10

    August 13

    • Started pDawn ClyA & pDawn ClyA-RFP cultures for blood agar experiment
    • Meeting with Dr. Sarkar to discuss progress on both projects

    August 14

      • Reviewed sequencing results and transformed the following into BL21 and DH5 alpha:
      • pET26b-INPNC
      • pET26b-INPNC-HA
      • pET26b-6xHis-DARPin-HA
      • pET26b-INPNC-DARPin-HA
      • pET26b-ClyA-6xHis
      • pET26b-PelB-ClyA-6xHis
      • pDawn-ClyA-6xHis
    • Serial dilution and plating of pDawn ClyA & pDawn ClyA-RFP cultures for blood agar experiment

    Week 11

    August 15

    • Re-plated all constructs
    • pDawn-ClyA and pDawn-ClyA-RFP demonstrated light-dependent hemolysis!
    • BL21 washing titration experiment for flow cytometry experiment - obtained optimal starting OD600 of 0.05 to obtain ~1E6 cells after all washes and incubations
    • Made Incubation Buffer for flow cytometry experiment
    • Designed/ordered pBAD33-eGFP Primers

    August 16

    • Picked colonies on all BL21 and DH5 constructs, including inducing and non-inducing conditions, set up flow cytometry stuff, booked flow core
    • Started cultures of pDawn-ClyA, pDawn-ClyA-RFP, and pDawn-ClyA-mCherry for repeat blood agar experiment
    • Set up light incubator properly
    • Started protein purification cultures of pDawn-ClyA-6xHis, pET26b-ClyA-6xHis, and pET26b-PelB-ClyA-6xHis, grew to OD600=0.8, induced with IPTG or light

    August 22

    • Image INPNC-DARPin-HA, INPNC-HA, DARPin-HA (both induced and not induced) on the confocal - Avin
    • Figure out ClyA imaging system and maybe design a stencil (not a high priority)
    • Pick up media and everything else from cell center - Mike
    • Ligate ClyA into lactococcus? -- Talk to Daphne about vectors
    • Order the cytotoxicity assay from Promega - Avin
    • Ask Dr. Sarkar for cell center account - Mike
    • Order pDawn sequencing primer - Avin
    • Run GFP PCR product on Gel, Gel didn’t work → rerun PCR using gradient annealing temp Run PCR products on gel, check if works
    • Look into biobrick format - Mike
    • Make an in-depth plan for the incoming weeks, prioritizing what needs to be done Cloning! what is the final construct? Design and execute add RBS to pBAD33 primer

    Plan for August 23

    • Add RBS/SD to pBAD33 forward primer and re-order it
    • Send out sequencing for INPNC-DARPin-HA (and what else?)
    • Look at old sequencing data just to make sure everything worked
    • Pick up IPTG from the cell center and make stock solution/aliquots
    • Using new IPTG, spread on blood agar plates (final dilution of 1 to 1000)
    • Spread Kan on Blood Agar Plates
    • Plate pET26b-ClyA-His (+/-), pET26b-pelB-ClyA-His (+/-), and pDawn-ClyA(FS) (+/-) on Blood Agar + Kan plates

    August 24

    • If colonies were picked, miniprep, then test cut, then run on diagnostic gel → transform into BL21
    • If colonies were not picked, then pick colonies
    • Analyze sequencing results
    • Plan out INPNC-DARPin-HA experiments
    • Figure out cloning for 1 plasmid system

    Week 12

    Goals in order of priority:

    • Show that INPNC-DARPin-HA binds to HER2 in vitro
    • Show that purified 6xHis-DARPin-HA binds to Her2 - Monday?
    • Show that INPNC-DARPin-HA is displayed on the surface
    • Show INPNC-mCherry is displayed on surface (confocal?)
    • Construct ClyA and ClyA-His BioBricks
    • Construct INPNC-mCherry Biobrick
    • Construct Surface Display BioBrick (need INPNC-mCherry to work)
    • Transform ClyA into Lactococcus and plate onto blood agar for Human Practices-
    • Stencil experiment showing spatial control of pDawn-ClyA (could be done next week, only takes a little time and artistic ability)
    • Show that cph8 works with a reporter
    • Show that cph8-ClyA works

    Experiments in order of priority:

    • Construction of pBAD33-eGFP (Mike)
    • Order primers for ClyA-His and ClyA to clone them into BioBrick backbone
    • Order primers for INPNC-mCherry
    • Order primers for cph8-reporter, cph8-clyA-his
    • SKBR3 imaging with pBAD33-eGFP/pET26b-INPNC-DARPin-HA bacteria on confocal
    • INPNC-DARPin immunos, 0.2mM IPTG, overnight induction
    • INPNC-DARPin bacterial flow cytometry on FACS machine (can use same bacteria as immuno)
    • Create stable cell line of SKBR3-mCherry (Jordan)
    • Obtain Lactococcus expression vector, order primers to clone in ClyA-His
    • Design surface display biobrick
    • Cytotoxicity experiment on SKBR3
    • Protein purification on His-DARPin-HA (Peter)
    • Flow cytometry of SKBR3 cells incubated with his-DARPin-HA (someone/Najaf)
    • Construct cph8-ClyA plasmid
    • cph8-reporter timecourse
    • cph8-ClyA blood agar

    August 27

    • PCR eGFP out of PHAT (annealing = 65), run on 1% gel, gel purify
    • Digest eGFP and pBAD33 with PstI and XmaI, column purify
    • Ligate at RT for 1 hour, transform into DH5a depending on time, otherwise ligate at 16C/4C overnight and transform the next day
    • Order Primers for (in order of priority)
    • ClyA and ClyA-His Biobricks
    • INPNC-mCherry construct
    • ClyA into lactococcus plasmid
    • Cph8-reporter plasmid
    • Cph8-ClyA-His plasmid
    • INPNC surface display vector (provided it works)
    • Start cell culture of SK-BR-3 cells?
    • Get protocols

    August 28

    • start cultures for purification of His-DARPin-HA
    • HA affinity purification of INPNC-DARPin-HA?
    • Print and hand in safety form to EHRS
    • Pick up SK-BR-3 plate from Cal, learn protocols
    • Add FBS to our McCoy’s media
    • Design and order primers to put ClyA into lactococcus expression vector
    • Set up preliminary stencil experiment, send avin plate base diameter (approximate 86mm)
    • Design INPNC-mCherry-DARPin primers
    • pBAD33-eGFP
    • Run Gradient PCR products (which ran overnight) on gel, gel purify the best band
    • Digest eGFP and pBAD33 with PstI and XmaI, column purify/gel purify
    • Ligate at RT for 1 hour, transform into DH5a depending on time, otherwise ligate at 16C/4C overnight and transform the next day
    • Start DH5 alpha cultures of INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin-HA
    • Also start cultures of pET-ClyA-His, pET-PelB-ClyA-His and pDawn-ClyA-His for minipreps

    August 29

    • Induce His-DARPin-HA culture (100 microliters of 1 M IPTG)
    • Monitor SK-BR-3
    • Primers for ClyA into lactococcus
    • Primers for INPNC-mCherry-DARPin
    • Pick colonies for pBAD33-eGFP if the RT ligation and transformation worked - Peter/Ashwin
    • If RT ligation and transformation looks bad, transform the 4C and 16C ligations - Mike
    • When primers arrive, PCR out mCherry from JMIL intein plasmid, PCR out INPNC, then assembly PCR for INPNC-mCherry

    August 30

    • Protein purification of His-DARPin-HA
    • PCR purify INPNC-mCherry, run a few ul on a diagnostic gel → results call for gel purifiation → Gel purify INPNC-mCherry → yield was bad, redo PCR → gel purify
    • Digest INPNC-mCherry and pET26b with NdeI and HindIII, ligate at RT for 1 hr (and maybe some at 4C or 16C overnight), and transform into pET26b
    • Start DH5a cultures for INPNC, INPNC-HA, His-DARPin-HA, pET-ClyA-His, pET-PelB-ClyA-His and pDawn-ClyA-His for minipreps
    • Repick colonies of pBAD33-eGFP from 16C or 4C plate

    September 2012 Notebook

    Week 13

    Week 14

    September 10

    • Mike - pick INPNC-mCherry colony (transformed Sample #2) and split into + and - cultures
    • Start BL21 cultures of INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin-HA, His-Intein-mCherry
    • Peter - Protein purification of His-DARPin-HA, ClyA-His
    • Avin - Dilute (9am) & Induce (?) His-DARPin-HA, INPNC-HA, INPNC-DARPin-HA at 0.5
    • Ashwin - Wiki work

    September 11

    • Mike - Make glycerol stock of INPNC-mCherry, Dilute and induce INPNC-mCherry, INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin HA, His-Intein-mCherry
    • Peter - Make electro/chemically competent cells, transform
    • Ashwin - Pick up S. typhimurium from Mark Goulian

    September 12

    • Peter - Lysis and spin down (for INPNC-mCherry, aliquot 1mL of both (+) and (-) for Avin
    • Peter- spin down and check if red, then lyse) , put in 4C, check Nissle eGFP colonies
    • Avin - Immuno experiment on His-DARPin-HA, INPNC-HA, INPNC-DARPin-HA, Take INPNC-mCherry, His-Intein-mCherry, fix and mount onto scope slides

    September 13

    • Mike - Bradford assay, Run protein gel
    • Peter -
    • Avin - Observe BL21 transformatons under confocal
    • Ashwin - colony PCR on S. typhimurium for MisL

    September 14

    • Mike -
    • Peter -
    • Avin - core confocal?
    • Ashwin -
      

    Other

    • Order scfv plasmid, scfv primers
    • Order MisL Primers, pick up misL, PCR on S. typhimurium
    • Chemical/Electrocompetent Nissle
    • Make LB, Glycerol
    • Wiki

    Week 15

    Week 16

    Retrieved from "http://2012.igem.org/Team:Penn/Notebook"