Team:Penn/Notebook

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  <img src="http://upload.wikimedia.org/wikipedia/en/d/d6/IGEM_official_logo.png" id="igem"/><!--igem logo-->
 
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<img src="http://collegediabetesnetwork.org/wp-content/uploads/2012/07/UPenn_logo1.png" id="penn"/> <!--penn logo-->
 
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                  <a href="https://2013.igem.org/Team:Penn/HumanPractices"><li>human practices</li></a>
 
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<a href="https://2013.igem.org/Team:Penn/Notebook"><li  class="active" style="background-color: rgb(200,200,215);">notebook</li></a>
 
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    <div class="section-title">notebook</div>
 
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        <li>Week 1</li>
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</div>
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        <li>Week 2</li>
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        <li>Week 3</li>
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        <li>Week 4</li>
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        <li>Week 5</li>
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        <li>Week 6</li>
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        <li>Week 7</li>
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        <li>Week 8</li>
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        <li>Week 9</li>
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        <li>Week 10</li>
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        <li>Week 11</li>
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        <li>Week 12</li>
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        <li>Week 13</li>
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        <li>Week 14</li>
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        <li>Week 15</li>
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        <li>Week 16</li>
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        <li>Week 17</li>
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        <li>Week 18</li>
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         <h2>WEEK 1</h2>
         <h2>WEEK 1</h2>
         <div class="box">
         <div class="box">
         <h2>June 4 2013 - June 11 2013</h2>
         <h2>June 4 2013 - June 11 2013</h2>
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         <img src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/APrj2Se2i-/Lynch%20Labs%20PGFI/BE_LynchLabGroup_DSC2811.jpg?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg" class="image"/><li>4-Jun
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         <img src="https://static.igem.org/mediawiki/2013/2/22/BE_LynchLabGroup_DSC2796.jpg"  width = "600px" class="image"/>
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             <ol>
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             <p><b>Goals:<br></b>
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            <li>Learned how to make competent cells, growing up two strains for tomorrow</li>
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<p>   This was our first week setting foot in the lab as the Penn iGEM team. We wanted to become more acquainted with the lab and start transforming and growing parts that would be useful in our future project ideas. This week we wanted to brainstorm our project ideas further as well as being more competent in basic molecular biology techniques.</p><br>
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            <li>Transformed 8 plasmids</li>
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<p><b>Achievements:<br></b>
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            <li>Determined EL222 fusion is risky but still going ahead with it</li>
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<p>We learned how to make competent cells and performed our first transformations of the summer. We continued to brainstorm potential projects and thought about directions we would like to pursue for the summer. Our idea for a project that would revolve around quorum sensing systems is currently being put into motion. We continued brainstorming for our idea about uses for DNA binding domains like TALE, Cas9, and zinc fingers. We grew up and miniprepped our transformations of various quorum sensing parts and learned how to use a plate reader.</p><br>
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            <li>Linkers are totally setlled</li>
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</div>
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            <li>Found zinc finger plasmid and updated target sequence</li>
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            <li>Learned how to make tetr- mcherry fusion</li>
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            <li>Settled on 5 promoters</li>
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      </ol></li>
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      <br/>
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        <li>5-Jun
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      <ol>
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            <li>Learned how to make competent cells, testing them and then making more tomorrow</li>
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            <li>Transformed 8 plasmids again</li>
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            <li>Made primers to clone the TET-GFP reporter system, the mCherry promoter strength system,
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ready to order</li>
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            <li>Made ultramers for variable promoter blocks (and no target neg controls) – ready to order</li>
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            <li>Spilled a lot of iced tea outside, bummer</li>
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            <li>Started primers for dna binding machines</li>
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            <li>Got a handle on cas9 fusions (pun intended).</li>
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            <li>Put awesome pics in dropbox</li>
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      </ol></li>
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      <br/>
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      <li>6-Jun
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            <ol>
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            <li>Clean up dropbox</li>
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            <li>Update budget sheet with addgene and cell center orders</li>
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            <li>Finish primers for fusion</li>
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            <li>Set up plate reader for GFP and mCherry assays</li>
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            <li>run minipreps on pdawn, pdawn-mcherry, pet26b</li>
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            <li>Grow up mCherry stock</li>
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            <li>Wrote Penn iGEM on our plasmid</li>       
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      </ol></li>
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      <br/>
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          <li>7-Jun
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      <ol>
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            <li>Transform</li>
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            <ol>
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                <li>C0012 –amp/chlor (do both)</li>
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                <li>M11307 – amp/chlor (do both)</li>
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                <li>I13458 – amp/chlor (do both)</li>
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                <li>R0010 – amp/chlor (do both)</li>
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                <li>R0051 – amp</li>
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                <li>K206000 –chlor</li>
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            </ol>
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            <li>Start the LIMS and file all the strains and DNA we have made/ ordered</li>
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      </ol></li>
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      <br/>
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          <li>8-Jun</li> <ol>
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            <li>Miniprep Addgene stuff + transformations that worked</li>
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            <li>Growing up low copy plasmids in 40mLs</li>
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      </ol>          <li>Mini-prepped</li><ol>
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                <li>I9002</li>
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                <li>I13458</li>
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                <li>C0051</li>
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                <li>Pdawn-mcherry</li>
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                <li>Pdawn</li>
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                            <li>Dhsa mcherry</li>
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                <li>Pdawn dhsa</li>
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                <li>Psb1a3</li>
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                <li>JM mcherry</li>
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                <li>Pet26b</li>
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            </ol>    </div>
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             <h2>June 11 2013 - June 18 2013</h2>
             <h2>June 11 2013 - June 18 2013</h2>
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         <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/-ZtRJ7Gu1U/photo-8.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
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         <img class="image" src="https://static.igem.org/mediawiki/2013/3/3e/Photo-8.JPG" width = "600px">
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            <li>17-Jun <ol>
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              <p><b>Goals:<br></b>
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            <li>Grow up luxI culture and grow up tetR culture </li>
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<p>   This week we wanted to transform some parts for our methylation idea that we ordered from addgene. We also wanted to attempt a bisulfite conversion to test methylation. We had some problems transforming some biobrick parts the previous week, so we wanted to troubleshoot our transformations as well. Another goal of ours was to make more competent DH5-alpha and Dam- cells as well as figure out methylation assays for our promoters.</p><br>
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            <li>Sequence all of the minipreps</li>
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<p><b>Achievements:<br></b>
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            <li>Transform t9002 in psb1A3 in NEB10</li>
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<p>We worked out the details of one of our potential projects: creating DNA binding domain-methyltransferase fusions. Like last week, we transformed and miniprepped parts that we would need for the quorum sensing project, like the LuxI system. We also transformed and miniprepped the standard biobrick backbones psb1A3, psb1C3, and psb1K3, which would be useful in almost any project we decide to pursue. We pinned down and ordered exactly the DNA binding domains that we thought we would be using and ordered the gene blocks from addgene. We methylated a biobrick part, c0051, with an M.sssI, and used our bisulfite conversion kit for the first time. </p><br> </div>
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            <li>Retransform ptetGFP to see if BL21DE3 cells are competent </li>
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            <li>Transform r0079, k081015, r0063 in NEB10 </li>
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            <li>Miniprep psb1k3</li>
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            <li>Redo dam gel with more dna </li>
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            <li>Figure out second control zfp from addgene </li>
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            <li>Figure out how to add luxR binding site to target region </li>
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            <li>Order sequencing primers for all addgene minipreps </li>
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            <li>Bisulfite converted msssi methylated c0051</li>
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      </ol></li>
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      <br/>
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      <li>18-Jun<ol>
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            <li>The bisulfite conversion was missing a negative control (bisulfite converted but unmethylated c0051) we'll need this to interperet results</li>
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            <li> Miniprep c0078, c0079 + make glycerol stocks check same plasmid with our kit </li>
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            <li>Order 13420 (second zfp)</li>
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<li>Design a way to make variable promoters more easily varied (for a biobrick so teams can use the reporter plasmid for their own nefarious reasons)</li>
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      </ol></li>
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      <li>19-Jun  <ol>
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            <li>Transform failed transformation</li>
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            <li>  Make competent DH5a && Dam- </li>
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            <li>Figure out methylation assays for promoters</li>
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            <li>Miniprep psb1A3 && all the 40mL cultures</li>
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            <li>Picked many colonies</li>
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            <li>Check pTet-gfp under blue light</li>
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      </ol></li> </div></div>
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         <h2>WEEK 3</h2>
         <h2>WEEK 3</h2>
         <div class="box"> <h2>June 18 2013 - June 25 2013</h2>
         <div class="box"> <h2>June 18 2013 - June 25 2013</h2>
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         <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
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         <img class="image" src="https://static.igem.org/mediawiki/2013/2/21/Aaathermopcrlala.jpg" width = "600px">
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<p><b>Goals:<br></b>
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<li>21-Jun<ol>
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<p>   It was increasingly important that we decide on our project. At this point we were considering a quorum sensing/cell signaling project that would build upon previously created quorum sensing parts submitted in the registry. We were also considering a methyltransferase-DNA binding domain fusion project with the goal of creating a new methyltransferase. We wanted to begin cloning t9002, a GFP producer controlled by an AHL signaling molecule receiver into psb1C3 from psb1A3 in order to use it in a quorum sensing device we were imagining.<br></b><p><b>Achievements:</b><br>
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        <li>troubleshoot plux-luxI pcr</li>
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<p>This week we got all of our minipreps submitted for sequencing, and we made headway on our first cloning reaction. We figured out how to add restriction sites for cloning reactions using PCR and completed our first cloning reaction according to our successful colony PCR. We experimented with different thermocycler conditions and polymerases for our PCRs that had previously failed, and began to perform PCR with more success.</p><br></div>
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        <li>roubleshoot pdawn-luxI pcr</li>
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        <li>made pDawn-tetR pcr work</li>
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        <li>troubleshoot pet26b-tetR pcr</li>
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        <li>troubleshoot pDawn-GFP pcr</li>
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        <li>troubleshoot pDawn-mCherry-secretion tag pcr </li>
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        <li>miniprep growing cultures, be sure to pick only the glowing ligations </li>
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        <li>ransform the correct t9002 amp ligation - determined from gel</li>
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        <li>digested t9002 in amp and ptet gfp in amp to identify the correct ligation</li>
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        <li>all of the chosen ptet gfp ligations worked, but let's not use 5 || 13 12. troubleshoot t9002 digest</li>
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        <li>troubleshoot t9002 digest</li><ol>
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            <li>check for contamination of something (run uncut sample, sample + buffer, sample + 1
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enzyme, sample + other enzyme, sample + both enzymes)</li>
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        </ol>
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      <br/>
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      <li>24-Jun<ol>
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        <li>Dam-/dh5a v dam methylation + dpnI && dpnII digest</li>
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        <li>Digested/ligated/transformed t9002 in amp</li>
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        <li>Get methylated biobrick sequenced</li>
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-
        <li>get chlor backbones sequenced</li>
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-
        <li>culture t9002 transformations in liquid media with i751250</li>
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        <li>mini prep stuff in the incubator</li>
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        <li>figure out the primer issues 8</li>
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        <li> Pick t9002 colonies for miniprep </li>
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      </ol></li></div>
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         <h2>WEEK 4</h2>
         <h2>WEEK 4</h2>
         <div class="box"> <h2>June 25 2013 - July 2 2013</h2>
         <div class="box"> <h2>June 25 2013 - July 2 2013</h2>
-
         <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/1LlS0hsVIZ/Screen%20shot%202013-05-29%20at%2016.22.13.png?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
+
         <img class="image" src="https://static.igem.org/mediawiki/2013/b/b3/Device_photo.jpg" width = "600px"><p><b>Goals:<br></b>
-
<ul><li>26-Jun<ol>
+
<p>   This week we wanted to think about applications for selective methylation in E. coli and figure out how our project could be useful for future research. We brainstormed how we wanted to create our constructs and began thinking about the cloning reactions that would be required to create them. We also wanted to begin creating our wiki and start generating useful content.<br></b><p><b>Achievements:</b></p>
-
        <li>Dam-/dh5a v dam methylation + dpnI && dpnII digest</li>
+
<p>We confirmed that we completed our first successful cloning reaction. We transformed and miniprepped the biobrick parts for the quorum sensing system that we envisioned creating, with one cell having a light-induced AHL producer, and another cell having a AHL producer downstream of an AHL activated promoter. We thought about creating a device that would spatially separate the cells, as opposed to having them in the same media. We used a simple restriction digest assay to test how BstUI, a methylation sensitive promoter, would behave. </p><br></div><!--/box-->
-
        <li>Digested/ligated/transformed t9002 in amp </li>
+
-
        <li>get chlor backbones sequenced</li>
+
-
        <li>culture t9002 transformations in liquid media with i751250 </li>
+
-
        <li>mini prep stuff in the incubator</li>
+
-
        <li>figure out the primer issues</li>
+
-
        <li> Pick t9002 colonies for miniprep</li>
+
-
        <li>USER Cloning reporter plasmid </li>
+
-
      </ol></li>
+
-
<br/>
+
-
   
+
-
        <li>1-Jul<ol>
+
-
            <li>Beautiful Brady Bunch photoshoot</li>
+
-
            <li>Troubleshooted and Re-tred PCR for user ends for reporter plasmid</li>
+
-
            <li>Called IDT about pcr assembly – they said Gibson tends to work better, no mutations, all in one tub. If we must PCR assembly – add DMSO, hotstart reaction, anneal at 68-70C (we did this).</li>
+
-
            <li>Get methylated biobrick sequenced </li>
+
-
            <li>Only sequence ptet GFP 11, if verified make sure to note on LIMS that we are only using ptet GFP 11 </li>
+
-
            <li> Check if plux/luxI system is working in liquid cultures – this failed</li> <ol style =""margin-left: 50px;"">
+
-
                <li>a. Might be strain competition, need to know growth rates</li>
+
-
            </ol>
+
-
            <li>Re-suspend primers for lux amplifier </li>
+
-
            <li>Mini-prep: e0040, psb1a3, r0062</li>    
+
-
        </ol></li>
+
-
        <br/>
+
-
       
+
-
        <li>2-Jul <ol><br>
+
-
            <li>Think about application of mathylation project in e.coli</li>
+
-
            <li>Ceck if plux/GFP-psb1C3 system is working in liquid cultures
+
-
            <ol>
+
-
                <li>+/- AHL induction at 100nM</li>
+
-
                <li>Compare with ptetGFP fluorescence, normal LB fluorescence</li>
+
-
            </ol></li>
+
-
            <li>Streak zinc finger 2</li>
+
-
            <li>Grow up 44251</li>
+
-
            <li>Transform up R0062</li>
+
-
            <li>When BstuI arrives<ol>
+
-
                <li>Assay BstuI working</li>
+
-
                <li>Assay the bvluc and tale1 minipreps in dh5a, dam-, and bl21 cells with msssi and bstuI </li>
+
-
                <li>Results: BstUI is blocked by methylation, and cells don’t normally methylate</li>
+
-
            </ol></li>
+
-
            <li>Growing up t9002 in chlor and i751250 in amp for fluorescence study</li>
+
-
            <li>Investigate CHIP or other ways of determining DNA binding domain specificity </li>
+
-
        </ol></li></ul></div><!--/box-->
+
     </div>
     </div>
      
      
Line 379: Line 76:
         <h2>WEEK 5</h2>
         <h2>WEEK 5</h2>
         <div class="box"> <h2>July 2 2013 - July 9 2013</h2>
         <div class="box"> <h2>July 2 2013 - July 9 2013</h2>
-
         <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Y5d0PLu26o/photo-7.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
+
         <img class="image" src="https://static.igem.org/mediawiki/2013/9/95/IMG_2175.JPG" width = "600px">
-
             <ul><li>3-Jul<ol>
+
             <p><b>Goals:<br></b>
-
            <li>Streak zinc finger 2 </li>
+
<p>   We wanted to experiment with USER cloning for assembling our gene blocks and minigenes. We also wanted to characterize pdawn and mcherry, parts we got from a lab at the University that we could use in our quorum sensing project.  We wanted to attempt to PCR assemble our MsssI and begin fabricating a device for our quorum sensing project. </p><br>
-
            <li>Grow up 44251 </li>
+
<p><b>Achievements:<br></b>
-
            <li>Look into lux box being light sensitive </li>
+
<p>We practiced measuring fluorescence using our pDawn and mcherry constructs. We successfully completed PCR assembly of our M.sssI. We performed our first cotransformation with I751250, an AHL sender that was downstream of pDawn and T9002. We redesigned primers for PCR reactions that had been giving us trouble, paying more attention to the annealing temperatures of the primers we were designing.</p><br></div>
-
        </ol></li>
+
-
       
+
-
        <li>4-Jul<ol>
+
-
            <li>Mini prep 44251 </li>
+
-
            <li>Pick ZFP2 colonies to grow up, throw out liquid culture in fridge </li>
+
-
        </ol></li>
+
-
       
+
-
        <li>5-Jul<ol>
+
-
            <li>Repeat BstUI assay, taking into account new controls</li>
+
-
            <li>Suspend the primers in the freezer </li>
+
-
            <li>We need to check if the origin of replications are compatible before co transformation</li>
+
-
            <li>Characterize pDawn-mcherry </li>
+
-
            <li>Practice measuring fluorescence</li>
+
-
        </ol></li>
+
-
       
+
-
       
+
-
        <li>6-Jul
+
-
      <ol>
+
-
            <li>troubleshoot ptetGFP user PCR - band was visible but too small to extract</li>
+
-
            <li>gel extract promoter fragments from USER PCR</li>
+
-
            <li>re-do USER PCR for: TetR, pTetGFP</li>
+
-
            <li>Design/check/order (Tale,Cas,ZFP)-flex-REV primers - check fwd primers</li>
+
-
        </ol></li></ul></div>
+
     </div>
     </div>
-
      
+
 
 +
<div>
 +
        <h2>WEEK 6</h2>
 +
        <div class="box"> <h2>July 10 2013 - July 17 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/b/b6/PennigemPic4.JPG" width = "600px">
 +
 
 +
        <p><b>Goals:<br></b>
 +
<p>    As the summer was nearing its halfway mark, we wanted to decide on the relative merits of the two projects and reach a decision. We also wanted to continue our cloning for the two projects and troubleshoot cloning-related problems. We now had all the parts we needed for Cas9, TALE, and zinc finger methylase constructs and wanted to begin assembling them into constructs that we envisioned. These constructs would have a two-plasmid system. One plasmid would have the DNA binding domain downstream of a T7 promoter activated by tetracycline, and the other would be a reporter plasmid to detect methylation. </p><br>
 +
<p><b>Achievements:<br></b>
 +
<p>We decided on the constructs that we would create and began the restriction digest and gel extract steps of our miniprepped components. We planned an experiment we could do with tetracycline induction to test whether our hypothetical methyltransferase-DNA binding domain fusions were actually being expressed.</p><br>
 +
      </div>
 +
     </div>
 +
<div>
 +
 
 +
<h2>WEEK 7</h2>
 +
        <div class="box"> <h2>July 18 2013 - July 24 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/6/61/Pennnotebook3pic.jpg" width = "600px">
 +
 
 +
        <p><b>Goals:<br></b>
 +
<p>    This week our main goals were to attempt our tetracycline induction experiment, order primers for performing a COBRA assay, and refine our LIMS and retroactively manage our samples. We also wanted to finish the cloning experiments we began the previous week.</p><br>
 +
<p><b>Achievements:<br></b>
 +
<p>We completed our construct for our zinc finger fusion plasmid after a successful colony PCR and sequencing result. We performed our first induction experiment by inoculating cultures with a tetracycline-activated promoter. We redid a PCR assembly of our TALE and Cas9 systems that had been failed before. </p><br>
 +
      </div>
 +
    </div>
 +
<div>
 +
 
 +
<h2>WEEK 8</h2>
 +
        <div class="box"> <h2>July 25 2013 - July 31 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/9/9e/IMG_2173.JPG" width = "600px">
 +
 
 +
        <p><b>Goals:<br></b>
 +
<p>    We wanted to finish our remaining fusion protein constructs and begin thinking about an SDS PAGE to measure protein expression. We also wanted to dedicate more time to pursuing drylab—making advances on our human practices and continue reading the literature on selective methylation. We also wanted to look into trying out different biobrick parts for our quorum sensing system.</p><br>
 +
<p><b>Achievements:<br></b>
 +
<p>We made advances on our cloning of the constructs for both projects. We completed a LIMS system on a Google spreadsheet that would be used to manage our increasingly overwhelming primers box as well as fixing our minipreps and glycerol stock LIMS and retroactively adding samples. We performed a new induction experiment, inducing our cultures with aTc. </p><br>
 +
      </div>
 +
    </div>
 +
<div>
 +
 
 +
<h2>WEEK 9</h2>
 +
        <div class="box"> <h2>Aug 1 2013 - Aug 7 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/5/59/IMG_0382.JPG" width = "600px">
 +
 
 +
        <p><b>Goals:<br></b>
 +
<p>    We wanted to make advances toward generating our device for the quorum sensing project. We wanted to better budget our remaining resources and stock up on supplies that we would need throughout the rest of the summer. We also wanted to use our zinc finger clone in an induction experiment, similar to the ones we had performed earlier for the quorum sensing project. Additionally, we aimed to generate a growth curve and see how the zinc finger cells would respond to induction. </p><br>
 +
<p><b>Achievements:<br></b>
 +
<p>We met with various professors at the University and made contacts who had knowledge about areas in both projects. We ordered more reagents and created the abstract for our project. We designed primers to attempt to Gibson assemble the Cas construct that had been giving us trouble during USER cloning. We hoped that the new cloning strategy would yield better results. We aliquoted our antibiotics, making boxes full of Kan, Chlor, and Amp to last us the entire summer and well into the year. </p><br>
 +
      </div>
 +
    </div>
 +
<div>
 +
 
 +
<h2>WEEK 10</h2>
 +
        <div class="box"> <h2>Aug 8 2013 - Aug 14 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/a/a5/IMG_0501.JPG" width = "600px"">
 +
 
 +
      <p><b>Goals:<br></b>
 +
<p>    We wanted to use our restriction digest-based test to analyze how our constructs were working with regards to methylation. We needed to decide on which project we were going to pursue as soon as possible, and we decided this week would be the deadline. We wanted to perform our first SDS Page, which had been in the brainstorming stage for a while. Additionally, we wanted to consult experts about designing better primers and see how we could improve our protocols. </p><br>
 +
<p><b>Achievements:<br></b>
 +
<p>We finally decided to fully pursue the project of constructing targeted methyltransferase fusions. While it was a hard decision, it was necessary to commit our efforts to one project. We also finished our TALE clone and began the induction experiment for testing the methyltransferase fusion. We consulted with designers from the School of Design about making our website and better presentations. We began transforming promoters for an additional screen for methyltransferases that we had envisioned, with the belief that methyltransferases could silence promoters.</p><br>
 +
      </div>
 +
    </div>
 +
<div>
 +
 
 +
<h2>WEEK 11</h2>
 +
        <div class="box"> <h2>Aug 15 2013 - Aug 21 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/b/be/Pennnotebook1pic.jpg" width = "600px" >
 +
 
 +
      <p><b>Goals:<br></b>
 +
<p>    We wanted to get our Cas construct working after additional drylab work in designing better assembly reactions. We needed to make a detailed plan of where our funds were going to go for the rest of the summer and begin planning our slides for our iGEM presentation. We wanted to decide where we wanted to be by the competition and what we needed to do to get there.</p><br>
 +
<p><b>Achievements:<br></b>
 +
<p>We began creating a plan for creating a video about methylation in general and our project specifically. After consulting with our advisors we decided to change the direction of our methyltransferase-DNA binding domain fusion from a two-plasmid to a one-plasmid system that would incorporate a digestion-based assay within the plasmid.</p><br>
 +
 
 +
      </div>
 +
    </div>
 +
<div>
 +
 
 +
<h2>WEEK 12</h2>
 +
        <div class="box"> <h2>Aug 22 2013 - Aug 28 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/b/bd/PennigemPic12.jpg" width = "600px">
 +
 
 +
      <p><b>Goals:<br></b>
 +
<p>    Our main goal for this week was to finalize our ideal cloning constructs and figure out how to port what we had already created into the one-plasmid system. We wanted to elaborate more on the assay, as our advisors had emphasized that as one of the more important contributions that our project could make to the synthetic biology community.</p><br>
 +
<p><b>Achievements:<br></b>
 +
<p>We focused on drylab this week, as we needed to get our plans for the rest of the summer set. We worked on a human practices essay about the ethics of targeted methylation in medical therapies. We also began work designing new primers for our revised cloning strategy.</p><br>
 +
      </div>
 +
    </div>
 +
<div>
 +
 
 +
<h2>WEEK 13</h2>
 +
        <div class="box"> <h2>Aug 29 2013 - Sep 4 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/f/f7/IMG_2180.JPG" width = "600px">
 +
 
 +
        <p><b>Goals:<br></b>
 +
<p>    Our goal was to once again begin a new cloning strategy focused on assembling our one-plasmid systems. We needed our cloning strategy finalized as soon as possible, and we dedicated the majority of our time this week to streamlining our cloning process.</p><br>
 +
<p><b>Achievements:<br></b>
 +
<p>We began setting up our travel plans to get to the Jamboree. We began the initial stages of cloning our new systems and made substantial progress on cloning our zinc finger and TALE. </p><br>
 +
      </div>
 +
    </div>
 +
<div>
 +
 
 +
<h2>WEEK 14</h2>
 +
        <div class="box"> <h2>Sep 5 2013 - Sep 11 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/a/ad/IMG_0495.JPG" width = "600px"">
 +
 
 +
        <p><b>Goals:<br></b>
 +
<p>  Our goal for this week was to make further progress on cloning our constructs. We also wanted to continue our work on the human practices essay, and begin creating a poster presentation for presenting at University research conferences. We also wanted to begin thinking about designing a computer application for analyzing gels.</p><br>
 +
<p><b>Achievements:<br></b>
 +
<p>During a marathon weekend coding session, we created a program that we called MaGellin that was designed to analyze our gels and characterize the amount of methylation. We attempted to improve our cloning efficiency by utilizing Antarctic Phosphatase within our reactions. We made substantial progress on finishing our TALE and zinc finger constructs, and selected and grew colonies that would be used to test our assay the following week. </p><br>
 +
      </div>
 +
    </div>
 +
<div>
 +
 
 +
<h2>WEEK 15</h2>
 +
        <div class="box"> <h2>Sep 12 2013 - Sep 18 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/d/d0/Pennigempic1.jpg" width = "600px"">
 +
 
 +
      <p><b>Goals:<br></b>
 +
<p>  We wanted to make substantial progress on the website this week. We also wanted to run our assay and test how well our methyltransferase-DNA binding domain fusions had worked. We wanted to work on animation for our video as well as make progress on our human practices essay.</p><br>
 +
<p><b>Achievements:<br></b>
 +
<p>We ran the assay on the TALE and zinc finger clones, and it yielded promising results. The expression of our fusion protein was a definite achievement. We made substantial progress on the website and essay as well. </p><br>
 +
      </div>
 +
    </div>
 +
<div>
 +
 
 +
<h2>WEEK 16</h2>
 +
        <div class="box"> <h2>Sep 19 2013 - Sep 25 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/5/5e/DSC_0482.JPG" width = "600px">
 +
 
 +
        <p><b>Goals:<br></b>
 +
<p>  We needed to finish our website and various forms this week, as well as run our assay in triplicate on the constructs we had created thus far. We needed to plan our remaining time up to the Jamboree, and practice our presentation skills at various outreach events.</p><br>
 +
<p><b>Achievements:<br></b>
 +
<p>We attended several events aimed at recruiting future team members that showed substantial interest in our research and iGEM in general. Our assay returned promising results about the state of our methyltransferase-DNA binding domain fusions. Additionally, we finished the website and filled out all the forms we needed. We have a few experiments still in progress, but our time now is spent preparing for the Jamboree and being ready to present our results. </p><br>
 +
      </div>
 +
    </div>
 +
<div>
 +
 
 +
 
 +
<h2>WEEK 17</h2>
 +
        <div class="box"> <h2>Sep 26 2013 - Oct 2 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/c/c8/Momomomomomomo.JPG" width = "600px">
 +
 
 +
      <p><b>Goals:<br></b>
 +
<p>  In the week before the Jamboree, we wanted to compile all our last-minute data as well as create our poster and Powerpoint presentation for the competition.</p><br>
 +
<p><b>Achievements:<br></b>
 +
<p>We gathered a ton of last-minute data this week. The solid deadline put upon us by the Jamboree motivated us all to work as hard as possible to get data worthy of our presentation at the Jamboree. While there's still a lot that we wish to accomplish with our project, we're proud of how far we've made it and hope our hard work pays off at the Jamboree. </p><br>
 +
      </div>
 +
    </div>
 +
<div>
 +
 
 +
<h2>WEEK 18 (North America Regionals)</h2>
 +
        <div class="box"> <h2>Oct 3 2013 - Oct 9 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/3/31/Pennigempic3.jpg" width = "600px">
 +
 
 +
      <p><b>Goals:<br></b>
 +
<p>  We just became the Regional Winners for North America for iGEM 2013, but there's no time to rest. Now it's time to get back in the lab and prepare for the World Jamboree. We want to make a game plan for what we are going to accomplish before the World Jamboree at MIT in early November and gather data in areas where we needed more work. We're taking the judge's comments from the regional Jamboree very seriously and attempting to improve our project as much as possible in the coming month. </p><br>
 +
<p><b>Achievements:<br></b>
 +
<p>We ran our MaGellin assay in a preliminary screening of our cas-methylase fusion and got some activity. We re-ran some previous experiments in triplicate to make the data more compelling. </p><br>
 +
      </div>
 +
    </div>
 +
<div>
 +
<h2>WEEK 19 </h2>
 +
        <div class="box"> <h2>Oct 10 2013 - Oct 16 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/b/b3/Pennnotebook2pic.jpg" width = "600px">
 +
 
 +
      <p><b>Goals: <br></b>
 +
<p>  We wanted to look further into the possibility of transcriptional silencing using targeted methylation. Additionally, we planned to run the full assay to screen our potential Cas-methylase fusion. Another major priority is to perform bisulfite sequencing to look at the target site for our TALE methylase. </p><br>
 +
<p><b>Achievements:  <br></b>
 +
<p>We continued to reach out to underclassmen and recruit members for the upcoming team. At the same time, we were hard at work in lab, performing our assay and trying to further quantify the extent of methylation in our fusion constructs. We got our bisulfite sequencing for our TALE submitted and are eagerly awaiting the results. </p><br>
 +
      </div>
 +
    </div>
 +
<div>
 +
<h2>WEEK 20</h2>
 +
        <div class="box"> <h2>Oct 17 2013 - Oct 23 2013</h2>
 +
        <img class="image" src="https://static.igem.org/mediawiki/2013/0/08/PennigemPic11.JPG" width = "600px">
 +
 
 +
      <p><b>Goals:<br></b>
 +
<p>  This week we wat to begin utilizing a cell-free system to test our targeted TALE methylase. It is imperative that we give the Wiki a complete design overhaul. We also want to make a plan for the couple weeks. </p><br>
 +
<p><b>Achievements:<br></b>
 +
<p> This week we were screening our cas methylase and found noticeable activity when done in triplicate with all the proper controls. We experimented with various induction times as well. We re-ran some experiments that had previous flaws or were missing controls in triplicate. We had some additional cloning to do to modify our constructs as well. We gathered data for our 3D plot of induction for our methylase fusion, which was perhaps the most time-consuming experiment we have done all year. </p><br>
 +
      </div>
 +
    </div>
 +
<div>
 +
      </div>
 +
    </div>
 +
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<h2>WEEK 21</h2>
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        <div class="box"> <h2>Oct 24 2013 - Oct 30 2013</h2>
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      <p><b>Goals:<br></b>
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<p>  Our main goal is to finalize Wiki for World Jamboree and to collect data for any major experiments we still need to wrap up. We also need to begin our poster for the Jamboree and work on our presentation. At the same time, there is a ton of wetlab and experiments that we have planned, all of which are unlikely to get done in the next couple weeks, but that won't stop us from trying. We have several presentations with various labs at Penn, including the epigenetics department this week.  </p><br>
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<p><b>Achievements:<br></b>
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<p>This week we finished the Wiki for World Competition. Additionally, we began our last set of experiments, for which the data will be revealed at the World Jamboree.  </p><br>
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<center><a href = "https://2013.igem.org/Team:Penn"> Home  </a> <a href = "https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf" >Spec Sheet</a> <a href = "https://2013.igem.org/Team:Penn/sitemap" >Sitemap</a>
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Latest revision as of 02:08, 29 October 2013

Penn iGEM

WEEK 1

June 4 2013 - June 11 2013

Goals:

This was our first week setting foot in the lab as the Penn iGEM team. We wanted to become more acquainted with the lab and start transforming and growing parts that would be useful in our future project ideas. This week we wanted to brainstorm our project ideas further as well as being more competent in basic molecular biology techniques.


Achievements:

We learned how to make competent cells and performed our first transformations of the summer. We continued to brainstorm potential projects and thought about directions we would like to pursue for the summer. Our idea for a project that would revolve around quorum sensing systems is currently being put into motion. We continued brainstorming for our idea about uses for DNA binding domains like TALE, Cas9, and zinc fingers. We grew up and miniprepped our transformations of various quorum sensing parts and learned how to use a plate reader.


WEEK 2

June 11 2013 - June 18 2013

Goals:

This week we wanted to transform some parts for our methylation idea that we ordered from addgene. We also wanted to attempt a bisulfite conversion to test methylation. We had some problems transforming some biobrick parts the previous week, so we wanted to troubleshoot our transformations as well. Another goal of ours was to make more competent DH5-alpha and Dam- cells as well as figure out methylation assays for our promoters.


Achievements:

We worked out the details of one of our potential projects: creating DNA binding domain-methyltransferase fusions. Like last week, we transformed and miniprepped parts that we would need for the quorum sensing project, like the LuxI system. We also transformed and miniprepped the standard biobrick backbones psb1A3, psb1C3, and psb1K3, which would be useful in almost any project we decide to pursue. We pinned down and ordered exactly the DNA binding domains that we thought we would be using and ordered the gene blocks from addgene. We methylated a biobrick part, c0051, with an M.sssI, and used our bisulfite conversion kit for the first time.


WEEK 3

June 18 2013 - June 25 2013

Goals:

It was increasingly important that we decide on our project. At this point we were considering a quorum sensing/cell signaling project that would build upon previously created quorum sensing parts submitted in the registry. We were also considering a methyltransferase-DNA binding domain fusion project with the goal of creating a new methyltransferase. We wanted to begin cloning t9002, a GFP producer controlled by an AHL signaling molecule receiver into psb1C3 from psb1A3 in order to use it in a quorum sensing device we were imagining.

Achievements:

This week we got all of our minipreps submitted for sequencing, and we made headway on our first cloning reaction. We figured out how to add restriction sites for cloning reactions using PCR and completed our first cloning reaction according to our successful colony PCR. We experimented with different thermocycler conditions and polymerases for our PCRs that had previously failed, and began to perform PCR with more success.


WEEK 4

June 25 2013 - July 2 2013

Goals:

This week we wanted to think about applications for selective methylation in E. coli and figure out how our project could be useful for future research. We brainstormed how we wanted to create our constructs and began thinking about the cloning reactions that would be required to create them. We also wanted to begin creating our wiki and start generating useful content.

Achievements:

We confirmed that we completed our first successful cloning reaction. We transformed and miniprepped the biobrick parts for the quorum sensing system that we envisioned creating, with one cell having a light-induced AHL producer, and another cell having a AHL producer downstream of an AHL activated promoter. We thought about creating a device that would spatially separate the cells, as opposed to having them in the same media. We used a simple restriction digest assay to test how BstUI, a methylation sensitive promoter, would behave.


WEEK 5

July 2 2013 - July 9 2013

Goals:

We wanted to experiment with USER cloning for assembling our gene blocks and minigenes. We also wanted to characterize pdawn and mcherry, parts we got from a lab at the University that we could use in our quorum sensing project. We wanted to attempt to PCR assemble our MsssI and begin fabricating a device for our quorum sensing project.


Achievements:

We practiced measuring fluorescence using our pDawn and mcherry constructs. We successfully completed PCR assembly of our M.sssI. We performed our first cotransformation with I751250, an AHL sender that was downstream of pDawn and T9002. We redesigned primers for PCR reactions that had been giving us trouble, paying more attention to the annealing temperatures of the primers we were designing.


WEEK 6

July 10 2013 - July 17 2013

Goals:

As the summer was nearing its halfway mark, we wanted to decide on the relative merits of the two projects and reach a decision. We also wanted to continue our cloning for the two projects and troubleshoot cloning-related problems. We now had all the parts we needed for Cas9, TALE, and zinc finger methylase constructs and wanted to begin assembling them into constructs that we envisioned. These constructs would have a two-plasmid system. One plasmid would have the DNA binding domain downstream of a T7 promoter activated by tetracycline, and the other would be a reporter plasmid to detect methylation.


Achievements:

We decided on the constructs that we would create and began the restriction digest and gel extract steps of our miniprepped components. We planned an experiment we could do with tetracycline induction to test whether our hypothetical methyltransferase-DNA binding domain fusions were actually being expressed.


WEEK 7

July 18 2013 - July 24 2013

Goals:

This week our main goals were to attempt our tetracycline induction experiment, order primers for performing a COBRA assay, and refine our LIMS and retroactively manage our samples. We also wanted to finish the cloning experiments we began the previous week.


Achievements:

We completed our construct for our zinc finger fusion plasmid after a successful colony PCR and sequencing result. We performed our first induction experiment by inoculating cultures with a tetracycline-activated promoter. We redid a PCR assembly of our TALE and Cas9 systems that had been failed before.


WEEK 8

July 25 2013 - July 31 2013

Goals:

We wanted to finish our remaining fusion protein constructs and begin thinking about an SDS PAGE to measure protein expression. We also wanted to dedicate more time to pursuing drylab—making advances on our human practices and continue reading the literature on selective methylation. We also wanted to look into trying out different biobrick parts for our quorum sensing system.


Achievements:

We made advances on our cloning of the constructs for both projects. We completed a LIMS system on a Google spreadsheet that would be used to manage our increasingly overwhelming primers box as well as fixing our minipreps and glycerol stock LIMS and retroactively adding samples. We performed a new induction experiment, inducing our cultures with aTc.


WEEK 9

Aug 1 2013 - Aug 7 2013

Goals:

We wanted to make advances toward generating our device for the quorum sensing project. We wanted to better budget our remaining resources and stock up on supplies that we would need throughout the rest of the summer. We also wanted to use our zinc finger clone in an induction experiment, similar to the ones we had performed earlier for the quorum sensing project. Additionally, we aimed to generate a growth curve and see how the zinc finger cells would respond to induction.


Achievements:

We met with various professors at the University and made contacts who had knowledge about areas in both projects. We ordered more reagents and created the abstract for our project. We designed primers to attempt to Gibson assemble the Cas construct that had been giving us trouble during USER cloning. We hoped that the new cloning strategy would yield better results. We aliquoted our antibiotics, making boxes full of Kan, Chlor, and Amp to last us the entire summer and well into the year.


WEEK 10

Aug 8 2013 - Aug 14 2013

Goals:

We wanted to use our restriction digest-based test to analyze how our constructs were working with regards to methylation. We needed to decide on which project we were going to pursue as soon as possible, and we decided this week would be the deadline. We wanted to perform our first SDS Page, which had been in the brainstorming stage for a while. Additionally, we wanted to consult experts about designing better primers and see how we could improve our protocols.


Achievements:

We finally decided to fully pursue the project of constructing targeted methyltransferase fusions. While it was a hard decision, it was necessary to commit our efforts to one project. We also finished our TALE clone and began the induction experiment for testing the methyltransferase fusion. We consulted with designers from the School of Design about making our website and better presentations. We began transforming promoters for an additional screen for methyltransferases that we had envisioned, with the belief that methyltransferases could silence promoters.


WEEK 11

Aug 15 2013 - Aug 21 2013

Goals:

We wanted to get our Cas construct working after additional drylab work in designing better assembly reactions. We needed to make a detailed plan of where our funds were going to go for the rest of the summer and begin planning our slides for our iGEM presentation. We wanted to decide where we wanted to be by the competition and what we needed to do to get there.


Achievements:

We began creating a plan for creating a video about methylation in general and our project specifically. After consulting with our advisors we decided to change the direction of our methyltransferase-DNA binding domain fusion from a two-plasmid to a one-plasmid system that would incorporate a digestion-based assay within the plasmid.


WEEK 12

Aug 22 2013 - Aug 28 2013

Goals:

Our main goal for this week was to finalize our ideal cloning constructs and figure out how to port what we had already created into the one-plasmid system. We wanted to elaborate more on the assay, as our advisors had emphasized that as one of the more important contributions that our project could make to the synthetic biology community.


Achievements:

We focused on drylab this week, as we needed to get our plans for the rest of the summer set. We worked on a human practices essay about the ethics of targeted methylation in medical therapies. We also began work designing new primers for our revised cloning strategy.


WEEK 13

Aug 29 2013 - Sep 4 2013

Goals:

Our goal was to once again begin a new cloning strategy focused on assembling our one-plasmid systems. We needed our cloning strategy finalized as soon as possible, and we dedicated the majority of our time this week to streamlining our cloning process.


Achievements:

We began setting up our travel plans to get to the Jamboree. We began the initial stages of cloning our new systems and made substantial progress on cloning our zinc finger and TALE.


WEEK 14

Sep 5 2013 - Sep 11 2013

Goals:

Our goal for this week was to make further progress on cloning our constructs. We also wanted to continue our work on the human practices essay, and begin creating a poster presentation for presenting at University research conferences. We also wanted to begin thinking about designing a computer application for analyzing gels.


Achievements:

During a marathon weekend coding session, we created a program that we called MaGellin that was designed to analyze our gels and characterize the amount of methylation. We attempted to improve our cloning efficiency by utilizing Antarctic Phosphatase within our reactions. We made substantial progress on finishing our TALE and zinc finger constructs, and selected and grew colonies that would be used to test our assay the following week.


WEEK 15

Sep 12 2013 - Sep 18 2013

Goals:

We wanted to make substantial progress on the website this week. We also wanted to run our assay and test how well our methyltransferase-DNA binding domain fusions had worked. We wanted to work on animation for our video as well as make progress on our human practices essay.


Achievements:

We ran the assay on the TALE and zinc finger clones, and it yielded promising results. The expression of our fusion protein was a definite achievement. We made substantial progress on the website and essay as well.


WEEK 16

Sep 19 2013 - Sep 25 2013

Goals:

We needed to finish our website and various forms this week, as well as run our assay in triplicate on the constructs we had created thus far. We needed to plan our remaining time up to the Jamboree, and practice our presentation skills at various outreach events.


Achievements:

We attended several events aimed at recruiting future team members that showed substantial interest in our research and iGEM in general. Our assay returned promising results about the state of our methyltransferase-DNA binding domain fusions. Additionally, we finished the website and filled out all the forms we needed. We have a few experiments still in progress, but our time now is spent preparing for the Jamboree and being ready to present our results.


WEEK 17

Sep 26 2013 - Oct 2 2013

Goals:

In the week before the Jamboree, we wanted to compile all our last-minute data as well as create our poster and Powerpoint presentation for the competition.


Achievements:

We gathered a ton of last-minute data this week. The solid deadline put upon us by the Jamboree motivated us all to work as hard as possible to get data worthy of our presentation at the Jamboree. While there's still a lot that we wish to accomplish with our project, we're proud of how far we've made it and hope our hard work pays off at the Jamboree.


WEEK 18 (North America Regionals)

Oct 3 2013 - Oct 9 2013

Goals:

We just became the Regional Winners for North America for iGEM 2013, but there's no time to rest. Now it's time to get back in the lab and prepare for the World Jamboree. We want to make a game plan for what we are going to accomplish before the World Jamboree at MIT in early November and gather data in areas where we needed more work. We're taking the judge's comments from the regional Jamboree very seriously and attempting to improve our project as much as possible in the coming month.


Achievements:

We ran our MaGellin assay in a preliminary screening of our cas-methylase fusion and got some activity. We re-ran some previous experiments in triplicate to make the data more compelling.


WEEK 19

Oct 10 2013 - Oct 16 2013

Goals:

We wanted to look further into the possibility of transcriptional silencing using targeted methylation. Additionally, we planned to run the full assay to screen our potential Cas-methylase fusion. Another major priority is to perform bisulfite sequencing to look at the target site for our TALE methylase.


Achievements:

We continued to reach out to underclassmen and recruit members for the upcoming team. At the same time, we were hard at work in lab, performing our assay and trying to further quantify the extent of methylation in our fusion constructs. We got our bisulfite sequencing for our TALE submitted and are eagerly awaiting the results.


WEEK 20

Oct 17 2013 - Oct 23 2013

Goals:

This week we wat to begin utilizing a cell-free system to test our targeted TALE methylase. It is imperative that we give the Wiki a complete design overhaul. We also want to make a plan for the couple weeks.


Achievements:

This week we were screening our cas methylase and found noticeable activity when done in triplicate with all the proper controls. We experimented with various induction times as well. We re-ran some experiments that had previous flaws or were missing controls in triplicate. We had some additional cloning to do to modify our constructs as well. We gathered data for our 3D plot of induction for our methylase fusion, which was perhaps the most time-consuming experiment we have done all year.


WEEK 21

Oct 24 2013 - Oct 30 2013

Goals:

Our main goal is to finalize Wiki for World Jamboree and to collect data for any major experiments we still need to wrap up. We also need to begin our poster for the Jamboree and work on our presentation. At the same time, there is a ton of wetlab and experiments that we have planned, all of which are unlikely to get done in the next couple weeks, but that won't stop us from trying. We have several presentations with various labs at Penn, including the epigenetics department this week.


Achievements:

This week we finished the Wiki for World Competition. Additionally, we began our last set of experiments, for which the data will be revealed at the World Jamboree.


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