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| | <style> | | <style> |
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| | + | /*Last year's notbook style code*/ |
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| | </head> | | </head> |
| | <body> | | <body> |
| | + | <div class="outer"> |
| | + | <ul class="nav"> |
| | + | <li><a href="https://2013.igem.org/Team:UPenn">Home</a></li> |
| | + | <li><a hred="https://2013.igem.org/Team:UPenn#three">Team</a></li> |
| | + | <li>Official Team Profile</li> |
| | + | <li><a href="https://2013.igem.org/Team:UPenn/Project">Project</a></li> |
| | + | <li><a href="https://2013.igem.org/Team:UPenn/Parts">Parts</a></li> |
| | + | <li><a href="https://2013.igem.org/Team:UPenn/Modeling">Modeling</a></li> |
| | + | <li><a href="https://2013.igem.org/Team:UPenn/Notebook">Notebook</a></li> |
| | + | <li><a href="https://2013.igem.org/Team:UPenn/Safety">Safety</a></li> |
| | + | |
| | + | </ul> |
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| | + | <iframe src='http://embed.verite.co/timeline/?source=0AoZBZOYYKBzEdDA1dUFNaU93QzQ4LURURjJfdzRiVFE&font=Bevan-PotanoSans&maptype=toner&lang=en&height=650' width='100%' height='650' frameborder='0'></iframe> |
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| | | | |
| - | Last year's notebook:
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| | <p style="text-align:center;color:white;">June 2012 Notebook</p> | | <p style="text-align:center;color:white;">June 2012 Notebook</p> |
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| | </ul> | | </ul> |
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| - | </div>
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| - | <h2 class="accordion-header">Week 4</h2>
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| - | <div class="accordion-content">
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| - | <p><b>June 22</b></p>
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| - | <p>Wet Lab</p>
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| - | <ul>
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| - | <li>Ashwin - Repeated miniprep on pDawn and did test cut</li>
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| - | <li>Peter - Miniprepped pet26b and digested with BglII and EcorRI</li>
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| - | <li>Avin - Miniprepped pet26b, made glycerol stock and digested with BamH1 and Not1</li>
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| - | </ul>
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| - | <p>Dry Lab</p>
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| - | <ul>
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| - | <li>Avin - Finalized and sent in synthesis order (still awaiting order confirmation)</li>
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| - | </ul>
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| - | <p><b>June 25</b></p>
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| - |
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| - | <p>Wet Lab</p>
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| - | <ul>
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| - | <li>Ashwin/Avin - Column purification, ligation, and transformation of Pet26b-mCherry</li>
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| - | <li>Peter -run gel of eGFP/plsr ligation</li>
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| - | </ul>
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| - |
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| - | <p>Dry Lab</p>
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| - | <ul>
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| - | <li>Avin - Sent in final gene synthesis order</li>
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| - | <li>Mike - reviewed pDawn protocol, reviewed TetR sequences</li>
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| - | <li>Peter- Order restriction enzymes from cell center</li>
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| - | </ul>
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| - | <br>
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| - | <p><b>June 26</b></p>
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| - | <p>Wet Lab</p>
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| - | <ul>
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| - | <li>Avin - Picked colonies for Pet26b-mCherry and pJT106b</li>
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| - | <li>Mike/Ashwin - Plated pJT122</li>
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| - | </ul>
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| - | <p>Dry Lab</p>
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| - | <ul>
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| - | <li>Everyone - Brainstormed human practices, Wiki design</li>
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| - | </ul>
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| - | <br>
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| - | <p><b>June 27</b></p>
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| - |
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <li>Miniprepped pet26b-mCherry and pDawn-mCherry</li>
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| - | <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)
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| - | Plated 200 ul and 20 ul</li>
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| - | <li>Inoculated colonies for pJT122, pJT106b, PhyB (BBa), PIF3 (BBa), and Cph8 (BBa)</li>
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| - | </ul>
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>Started exploring possible wiki designs and coding</li>
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| - | </ul>
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| - | <br>
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| - | <p><b>June 28</b></p>
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <li>Miniprepped pJT122, pJT106b, PhyB, PIF3, Cph8, made glycerol stocks</li>
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| - | <li>Picked colonies for pET26b-mCherry and pDawn-mCherry, innoculated, grew up, then diluted
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| - | Measured OD along the way</li>
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| - | </ul>
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>Set up light and dark incubators</li>
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| - | </ul>
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| - | <p><b>June 29</b></p>
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| - |
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <li>Tested pDawn-mCherry and pET-mCherry, pDawn-mCherry expressed mCherry only after light induction</li>
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| - | <li>Nanodropped pJT122, pJT106b, PhyB, PIF3, Cph8</li>
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| - | <li>Performed digest of pET-26b, eGFP, and plsr</li>
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| - | <li>Spread Biobrick shipment on Amp plates (lsrR, lsrK)</li>
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| - | </ul>
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| - |
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>Opened Bio-Rad shipments</li>
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| - | </ul>
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| - | <br>
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| - |
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| - |
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| - |
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| - | </div>
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| | </div> | | </div> |
| - | <p style="text-align:center; color:white;">July 2012 Notebook</p>
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| - | <div id="accordion-container">
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| - | <h2 class="accordion-header">Week 5</h2>
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| - |
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| - | <div class="accordion-content">
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| - |
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| - | <p><b>July 2</b></p>
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <li>Transformed ho1 and pcyA BioBricks</li>
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| - | <li>Peter: ligations</li>
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| - | </ul>
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| - |
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>Contacted labs for JT2 and pPL-PCB</li>
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| - | <li>Worked on Human Practices</li>
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| - | </ul>
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| - |
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| - | <br>
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| - |
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| - |
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| - |
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| - | <p><b>July 3</b></p>
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| - |
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| - | <p>Wet Lab</p>
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| - | <ul>
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| - | <li>Nothing to be done for drug delivery</li>
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| - | </ul>
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| - | <p>Dry Lab</p>
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| - | <ul>
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| - | <li>Avin - digital schematic, helped with primers, read human practices bacterial therapy stuff</li>
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| - | <li>Mike - talked to Dan, developed cloning strategy for getting Cph8 into the pDawn backbone, started primer design</li>
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| - | </ul>
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| - | </br>
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| - |
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| - |
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| - |
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| - | <p><b>July 5</b></p>
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| - |
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| - | <p>Wet Lab</p>
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| - | <ul>
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| - | <li>Picked colonies of ho1 and pcyA</li>
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| - | <li>Inoculated pDawn-mCherry cells for timecourse</li>
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| - | </ul>
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| - | <p>Dry Lab</p>
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| - | <ul>
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| - | <li>worked on primer design/cloning strategy and talked to Dan</li>
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| - | <li>worked on schematic and wiki</li>
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| - | <li>worked on human practices</li>
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| - | </ul>
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| - | </br>
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| - |
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| - | <p><b>July 6-7</b></p>
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| - |
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - |
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| - | <li>Took pDawn-mCherry time course</li>
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| - | <li>Sterilized incubator and TC hood</li>
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| - | <li>Moved lab stuff to small room</li>
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| - | <li>Redesigned primers for plsr & egfp</li>
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| - | </ul>
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| - |
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>
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| - | Contacted FDA contacts for human practices
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| - | </li>
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| - | </ul>
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| - | <br>
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| - |
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| - | </div>
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| - |
| |
| - | <h2 class="accordion-header">Week 6</h2>
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| - |
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| - | <div class="accordion-content">
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| - |
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| - | <p><b>July 9</b></p>
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| - |
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <li>Inoculated 50mL cultures of pDawn-mCherry</li>
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| - | <li>Set up pH meter, picked up JT2</li>
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| - | <li>Organized lab area </li>
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| - | <li>Made bacterial streaks for biobricks</li>
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| - | </ul>
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>Ordered mammalian cell culture media</li>
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| - | <li>Finalized wiki template</li>
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| - | <li>Worked on cloning steps for cph8</li>
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| - | </ul>
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| - | <br>
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| - |
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| - |
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| - |
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| - | <p><b>July 10</b></p>
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| - |
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <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>
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| - | <li>Picked colonies from transformed Biobricks and innoculated</li>
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| - | <li>Resuspended ClyA and LuxS IDT DNA and transformed into DH5alpha</li>
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| - | <li>TC/Sterile practice training</li>
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| - | </ul>
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| - |
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>Ordered primers for ClyA-RFP, Peters primers</li>
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| - | <li>Worked on wiki</li>
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| - | <li>Emailed Tabor for pJT106b plasmid map</li>
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| - | </ul>
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| - | <br>
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| - |
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| - |
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| - |
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| - | <p><b>July 11</b></p>
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <li>
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| - | Recorded BL21 pDawn-mCherry growth curve and calculated doubling time</li>
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| - | <li>
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| - | <ul> Miniprepped biobricks
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| - | <li>
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| - | BBa_K265008 - Ice Nucleation Protein NC
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| - | </li>
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| - | <li>
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| - | BBa_K523013 - Plac + INP-EYFP
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| - | </li>
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| - | <li>
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| - | BBa_K299810 - B0032 + Invasin
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| - | </li>
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| - | <li>
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| - | BBa_K257010 - ClyA + RFP
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| - | </li>
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| - | </ul>
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| - | </li>
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| - | </ul>
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| - |
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>Worked on biofilm project schematic</li>
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| - | <li>More work on wiki</li>
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| - | </ul>
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| - | <br>
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| - |
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| - |
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| - |
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| - | <p><b>July 13</b></p>
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| - |
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <li>Diluted to OD600=0.01 and take 0-8hr time points of pDawn-mCherry fluorescence time course</li>
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| - | <li>Thawed out HEK293T cells</li>
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| - | </ul>
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>Worked on wiki/schematic</li>
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| - | </ul>
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| - | <br>
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| - |
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| - | <p><b>July 17</b><p>
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - |
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| - | <li>Re-ligation of pDawn with IDTClyA and ClyA-RFP, Transformation into DH5alpha/XO1blue</li>
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| - | <li>Performed PCR of plsr & gfp w/new primers.</li>
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| - | <li>Picked colonies of T7 and INPNC-DARPin</li>
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| - | </ul>
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| - |
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>Designed experiments for ClyA and INPNC-DARPin assays</li>
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| - | </ul>
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| - | <br>
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| - |
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| - |
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| - | </div>
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| - |
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| - | <h2 class="accordion-header">Week 7</h2>
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| - |
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| - | <div class="accordion-content">
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| - |
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| - | <p><b>July 16</b></p>
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <li>
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| - | Transformed INPNC-DARPin and T7 BioBrick
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| - | </li>
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| - | </ul>
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| - |
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| - |
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>SAAST presentation</li>
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| - | <li>Met with Lazzara lab to discuss DARPin experiments</li>
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| - | </ul>
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| - | <br>
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| - |
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| - | <p><b>July 18</b></p>
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| - |
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <li>
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| - | Re-inoculated T7 and INPNC-DARPin</li>
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| - | <li>PCR ClyA-RFP, Gel purify PCR ClyA-RFP product</li>
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| - | </ul>
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| - |
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>Worked on DARPin assay experiment design</li>
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| - | </ul>
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| - | <br>
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| - |
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| - | <p><b>July 19</b></p>
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| - |
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <li>Trypan Blue Assay</li>
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| - | <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>
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| - | <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>
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| - | <li>Transformed all of above ligations into DH5alpha</li>
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| - | </ul>
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| - | <br>
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| - |
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| - |
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| - | <p><b>July 23</b></p>
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| - |
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <li>Ashwin-Design test cuts for pDawn-Clya, pDawn-ClyA+RFP </li>
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| - | <li>Ashwin-Digest pDawn-ClyA+pDawn-ClyA+RFP, Run on gel </li>
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| - | <li>Nikita - Miniprep pDawn culture in Sarkar cold room</li>
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| - | <li>Digest of pET-26b w/ BglII, EcoRI-Peter</li>
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| - | <li>Digest of plsr product w/ BglII, PseI-Peter</li>
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| - | <li>Digest egfp w/ SpeI, EcoRI-Peter</li>
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| - | <li>Ligate & transform pET-26-plsr-GFP-Peter</li>
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| - | <li>Digest INPNC-DARPin</li>
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| - | <li>Avin - Try to rescue 293T cells after CO2 arrives if can’t be rescued, thaw out more 293T</li>
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| - | <li>Transform pET-26b-luxS - Peter</li>
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| - | <li>Avin - pick GAVPO </li>
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| - | </ul>
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| - |
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>Ordered AI-2 ASAP-peter</li>
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| - | <li>Nikita - Researched whether ClyA is toxic to E. coli BL21, JT2, DH5 alpha, and whether it is secreted in these strains</li>
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| - | <li>Read Daphne+Dr. Sarkar’s ligation paper </li>
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| - | <li><ul>Avin - Buy:
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| - | <li>DH5 alpha</li>
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| - | <li>20 kan plates, 10 amp plates</li>
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| - | <li>Cell Counter</li>
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| - | </ul>
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| - | </li>
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| - | <li>
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| - | Write out competent cell production protocol-Peter</li>
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| - | <li>Obtained BL21 transformation protocol from daphne/find it-Peter</li>
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| - | <li>Avin - Logo design, human practices</li>
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| - | <li>Ashwin - wiki</li>
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| - | </ul>
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| - | </br>
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| - |
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| - |
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| - |
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| - | <p><b>July 24</b></p>
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| - |
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <li>Pick colonies of pDawn-ClyA and pDawn-ClyA+RFP (DH5alpha)</li>
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| - | <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>
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| - | <li>Avin - Ran Gel, no DNA</li>
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| - | </ul>
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| - |
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| - | <p>Dry Lab:</p>
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| - | <ul>
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| - | <li>Peter: order the promoter (or figure out an alternative method..)</li>
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| - | <li>Ashwin: wiki</li>
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| - | <li>Avin - Edit/Order primers for HA tag and His tag, design pDawn-ClyA experiments, human practices</li>
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| - | <li>Nikita - Write human practices</li>
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| - | </ul>
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| - | </br>
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| - |
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| - |
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| - | </div>
| |
| - | <h2 class="accordion-header">Week 8</h2>
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| - |
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| - | <div class="accordion-content">
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| - |
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| - | <p><b>July 26</b></p>
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| - |
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| - | <p>Wet Lab:</p>
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| - | <ul>
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| - | <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>
| |
| - |
| |
| - | <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>
| |
| - |
| |
| - | <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>
| |
| - |
| |
| - | <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>
| |
| - |
| |
| - | <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>
| |
| - |
| |
| - | <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">
| |
| - |
| |
| - | <p><b>August 1st</b></p>
| |
| - |
| |
| - | <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>
| |
| - | <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>
| |
| - |
| |
| - | <p>Dry Lab:</p>
| |
| - | <ul>
| |
| - | <li>Logo - Avin</li>
| |
| - | </ul>
| |
| - | <br>
| |
| - |
| |
| - | <p><b>August 3</b></p>
| |
| - |
| |
| - | <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>
| |
| - | <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>
| |
| - |
| |
| - | <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>
| |
| | | | |
| | | | |
"
