Team:Michigan/Notebook

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<!-- *** Here is where you should enter your lab notebook! Remember to name and date your entries. Use the icons on the top left corner to embed image, bold, italicize, and so forth.  For technical support, email huetone@gmail.com or ask Candy. *** -->
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<h2>Increasing Recombinase HbiF and FimE Turnover Rate with LVA Degradation Tag </h2>
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<p>HbiF and LVA degradation tag were ordered from IDT with the following specifications.</p>
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    <p><a href="/Team:Michigan/Project">Project</a> > <a href="/Team:Michigan/Noteboook">Notebook</a></p>
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<p> <span style="background-color:#E0771B;font-family:Courier, monospace;">EcoRI</span></p>
 
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<p> <span style="background-color:#E0B91B;font-family:Courier, monospace;">XbaI</span></p>
 
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<p> <span style="background-color:#C8E0DB;font-family:Courier, monospace;">SpeI</span></p>
 
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<p> <span style="background-color:#A9C9A4;font-family:Courier, monospace;">PstI</span></p>
 
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<p> <span style="background-color:#86C67C;font-family:Courier, monospace;">AgeI</span></p>
 
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<p> <span style="background-color:#CD96CD;font-family:Courier, monospace;">NgoMIV</span></p>
 
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<p><br/>HbiF possesses an N-terminus in the (truncated) RFC10 format :</p>
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<p style="font-family:Courier, monospace;"><span style="background-color:#E0771B">GAATTC</span> GCGGCCGC T <span style="background-color:#E0B91B;">TCTAG</span> [ATG]</p>
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<p><br/>No extra amino acids are created by this prefix, in comparison to the standard RFC25 prefix, which creates a serine and glycine. The suffix proceeds as follows:</p>
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<p style="font-family:Courier, monospace;"><span style="background-color:#86C67C;">ACCGGT</span> TAAT <span style="background-color:#C8E0DB">ACTAGT</span> A GCGGCCG <span style="background-color:#A9C9A4">CTGCAG</span> </p>
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<p><br/>In order to properly attach the LVA degradation tag, the prefix of the tag sequence must be complementary to the suffix of Hbif. For this part, the prefix and suffix are reversed in comparison to above: the prefix is RFC25, and the suffix is RFC10, for reasons that are discussed below.</p>
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<p><br/>Prefix:</p>
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<p style="font-family:Courier, monospace;"><span style="background-color:#E0771B;">GAATTC</span> GCGGCCGC T <span style="background-color:#E0B91B;">TCTAGA</span> TG <span style="background-color:#CD96CD;">GCCGGC</span></p>
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<p><br/>Suffix:</p>
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<p style="font-family:Courier, monospace;"><span style="color:red;">(TAA TAA)</span> T <span style="background-color:#C8E0DB;">ACTAGT</span> A GCGGCCG <span style="background-color:#A9C9A4;">CTGCAG</span></p>
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<p><br/>The suffix is RFC10 - we don’t need it to be RFC25 because two stop codons not in the original suffixes have been introduced before the suffix starts. LVA-mediated degradation depends on a series of amino acids at the C-terminus binding to cellular degradation machinery, and failure to abruptly halt transcription after this sequence may result in improper binding and degradation. Unlike in the RFC25 suffix, which adds linker amino acids after the protein domain ends, RFC10 adds a tyrosine before its internal stop codon, assuming translation proceeds that far - this could severely hinder binding.</p>
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    </div>
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<p class="title">Main tasks:</p>
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<h2>Cloning Team Notebook</h2>
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<iframe width=100% height=800; src="https://docs.google.com/document/pub?id=1bDaN8iqQjBHllPLosoQUs9Z9MZT4Q3pjOG3M_9UMEho&amp;embedded=true"></iframe>
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<ol>
 
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<li>Extract Hbif and the degradation tags from their parent plasmids, place in biobrick-compatible plasmids.</li>
 
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<li>Obtain RFC25-standard plasmid Bba-K157000 for use in construction.</li>
 
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<li>Clone the LVA tag onto Hbif’s C-terminus.</li>
 
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<li>Design primers to FimE which place an AgeI site at it’s C-terminus.</li>
 
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<li>Clone the LVA tag onto the modified FimE.</li>
 
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<li>Assay functionality of both. </li>
 
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</ol>
 
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<h2>Prepare FimE for protein expression</h2>
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<iframe width=100% height=500; src="https://docs.google.com/document/pub?id=1bDaN8iqQjBHllPLosoQUs9Z9MZT4Q3pjOG3M_9UMEho&amp;embedded=true"></iframe>
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</div>
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<h2>Team Reports</h2>
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<iframe src="https://docs.google.com/presentation/embed?id=17czITWHCoyzbzdYCvXWmwpDbl31jJyhgNmb32IXX3aY&start=false&loop=false&delayms=8000" frameborder="0" width=60% height=400 allowfullscreen="true" mozallowfullscreen="true" webkitallowfullscreen="true"></iframe>
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