Team:Penn/Notebook

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         <img src="https://static.igem.org/mediawiki/2013/2/22/BE_LynchLabGroup_DSC2796.jpg"  width = "600px" class="image"/>

             <p><b>Goals:<br></b>

<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>

<p><b>Achievements:<br></b>

<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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              <p><b>Goals:<br></b>

<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>

<p><b>Achievements:<br></b>

<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>

         <img class="image" src="https://static.igem.org/mediawiki/2013/2/21/Aaathermopcrlala.jpg" width = "600px">

<p><b>Goals:<br></b>

<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>

<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>

         <img class="image" src="https://static.igem.org/mediawiki/2013/b/b3/Device_photo.jpg" width = "600px"><p><b>Goals:<br></b>

<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>

<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-->

         <img class="image" src="https://static.igem.org/mediawiki/2013/9/95/IMG_2175.JPG" width = "600px">

             <p><b>Goals:<br></b>

<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>

<p><b>Achievements:<br></b>

<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>

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         <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>