http://2013.igem.org/wiki/index.php?title=Special:Contributions/Tycko&feed=atom&limit=50&target=Tycko&year=&month=2013.igem.org - User contributions [en]2024-03-28T22:12:38ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:Penn/TeamTeam:Penn/Team2014-01-15T16:43:57Z<p>Tycko: </p>
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<h1><b><center>About the Team</center></b></h1><br />
<div align="left">The Penn iGEM 2013 team is made up of 5 undergraduate students and various advisors and mentors at the University of Pennsylvania. The team comes from various academic disciplines such as math, bioengineering, and computer science. The team works out of the undergraduate bioengineering laboratory and is rumored to live there. <br />
<br><center><a href = 'https://docs.google.com/forms/d/188iefj_j6gBvrHC0IS3rG2zBelaVuy-JddYNN7QF8_Y/viewform'>Interested in joining the team? Submit your application for Penn iGEM 2014 here. </a></center><br />
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<h3>The Team Members</h3> <!--title of section--><br />
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<h2>Brad "Big Dark" Kaptur</h2><p>Bioengineering</p><br />
<p>"Screw minipreps, gotta get big."</p> <br />
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<h2>Josh Tycko</h2><p>Mathematics and Biology</p><br />
<p> "Don't worry, I know a guy."</p> <br />
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<h2>Danielle Fields</h2><p>Bioengineering</p><br />
<p>"I have a Starbucks Gold Card. Literally."</p> <br />
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<h2>Danny Cabrera</h2><p>Computational Biology</p><br />
<p>"Yo, [mumble] you pour [mumble] that gel yet?"</p> <br />
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<div class="member_divide"><h2>Mahamad "Dusty" Charawi</h2><p>Management and Computer Science</p><br />
<p>"Hi my name Mahamad, from Penn iGEM."</p> <br />
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<h3>The Advisors</h3> <!--title of section--><br />
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<div class="member_divide_text"><h2><a href='http://www.seas.upenn.edu/directory/profile.php?ID=178'>Brian Chow, Ph.D</a></h2><p>Assistant Professor</p><br />
<p>Chow's laboratory aims to create technological innovations to enhance therapeutic interventions in CNS disorders, and to this end, focuses on engineering tools to elucidate, diagnose and treat the diseased brain. He was instrumental in the direction of the team, teaching us science, organization, and leadership.</p> <br />
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<div class="member_divide_text"><h2>Spencer "STG" Glantz</h2><p>Bioengineering (PhD Student)</p><br />
<p>Spencer helped found the first iGEM team at the University of Pennsylvania and has been lending us his deep knowledge and experience with synthetic biology ever since. He helped us every day and truly made this project possible.</p> <br />
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<div class="member_divide_text"><h2><a href='http://www.orkantelhan.com/'>Orkan Telhan, Ph.D</a></h2><p>Assistant Professor</p><br />
<p> Interdisciplinary artist, designer, and researcher whose investigations focus on the design of interrogative objects, interfaces, and media, engaging with critical issues in social, cultural, and environmental responsibility. He greatly assisted the team with our efforts to properly communicate synthetic biology to the public.</p> <br />
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<div class="member_divide_text"><h2><a href='http://bioengineering.rice.edu/Content.aspx?id=4294967626'>Jordan Miller, Ph.D</a></h2><p>Assistant Professor of Bioengineering at Rice</p><br />
<p>Jordan has been an advisor to Penn iGEM teams for the last three years. He has given us insightful scientific advice and words of wisdom, and he sends the best emails.</p> <br />
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<div class="member_divide_text"><h2>Avin "Shiii" Veerakumar </h2><p>iGEM Guru</p><br />
<p>The Penn iGEM Godfather. He sent us great GIFs and drew a computer.</p> <br />
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<div class="member_divide_text"><h2>Michael Salvatore "Gumbaicci Margaricci" Magaraci</h2><p>iGEM Guru</p><br />
<p>Cloning Master. Diagram Master. He was a huge help.</p> <br />
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<h3>Nothing would be possible without these guys</h3> <!--title of section--><br />
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<div class="member_divide_text"><h2>Sevile Mannickarottu</h2><p>Director of Bioengineering Instructional Laboratories</p><br />
<p>The 2013 Penn iGEM team would like to thank Sevile for providing his facilities and equipment for use during the project.</p> <br />
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<div class="member_divide_text"><h2>Henry "HMFM" Ma </h2><p>Bioengineering (Masters Student)</p><br />
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<p>"Coffee?"</p> <br />
<p>Thank you to Henry for his wonderful support throughout this whole process.</p><br />
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<p style="text-align: center"><b>We could not have succeeded without the help of all these incredibly talented people. We are extremely grateful for their help.</b></p><br />
<p style="text-align: center">Generously donated interesting organisms and scientific expertise by <a href='http://www.bio.upenn.edu/people/mark-goulian'>Dr. Mark Goulian</a></p><br />
<p style="text-align: center">Advice on epigenetics by Drs <a href='http://www.med.upenn.edu/apps/faculty/index.php/g20000320/p13534'>Marisa Bartolomei</a> and <a href='http://www.med.upenn.edu/apps/faculty/index.php/g361/p11818'>Rebecca Simmons</a></p><br />
<p style="text-align: center">Advice on methylation assays by Chris Krapp</p><br />
<p style="text-align: center">Computer Vision algorithm by Eric Kauderer Abrams</p><br />
<p style="text-align: center">Graphic Design by Micah Kaats</p><br />
<p style="text-align: center">Assistance developing MaGellin user interface by Morgan Snyder</p><br />
<p style="text-align: center">Video editing and Animation by Dylan Petro</p><br />
<p style="text-align: center">Cinematography and Photography by Jake Whritner</p><br />
<p style="text-align: center">Music by Ben Brodie</p><br />
<p style="text-align: center">Photography by Erica Sacshe</p><br />
<p style="text-align: center">Web development by Dylan Petro and Stefanie Alfonso</p><br />
<p style="text-align: center">We would also like to thank <a href='http://davidgdula.com'>David Gdula</a> of NEB for being so personally invested in the team's success</p><br />
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</html></div>Tyckohttp://2013.igem.org/Team:PennTeam:Penn2014-01-15T16:42:39Z<p>Tycko: </p>
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<br><center><a href = 'https://docs.google.com/forms/d/188iefj_j6gBvrHC0IS3rG2zBelaVuy-JddYNN7QF8_Y/viewform'>Interested in joining the team? Submit your application for Penn iGEM 2014 here. </a></center><br />
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<center><object width="900" height="506"><param name="movie" value="//www.youtube.com/v/YJQniNpnU9Q?version=3&amp;hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="//www.youtube.com/v/YJQniNpnU9Q?version=3&amp;hl=en_US" type="application/x-shockwave-flash" width="800" height="506" allowscriptaccess="always" allowfullscreen="true"></embed></object></center><br />
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</TABLE><br><a href='https://2013.igem.org/Team:Penn/MaGellinToolbox'>Constructed a toolbox for developing site-specific DNA methylases</a></TD><br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/AssayOverview'><IMG SRC="http://farm3.staticflickr.com/2836/10529350575_dfb38e3538.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/AssayOverview'>Created a standardized assay for site-specific methylases</a></TD><br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/Software'><IMG SRC="http://farm4.staticflickr.com/3742/10529401844_cc5f713a48.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/Software'>Developed software to automate analysis of our assay</a></TD><br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/MethylaseOverview'><IMG SRC="http://farm6.staticflickr.com/5478/10529350195_2c54c496e5.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/MethylaseOverview'>Designed and characterized a novel TALE-methylase fusion protein</a> <br></TD><br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/hpoverview'><IMG SRC="http://farm4.staticflickr.com/3670/10529586493_833c6216ee.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/hpoverview'>Introduced synthetic biology to the community</a></TD><br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/Too_Soon_To_Treat'><IMG SRC="http://farm4.staticflickr.com/3815/10529365086_82d164e2b1.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/Too_Soon_To_Treat'>Published an article on the ethics of epigenetic engineering</a></TD><br />
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</TABLE><br><a href='https://2013.igem.org/Team:Penn/Team'>Our team</a> <br> <br> <br></TD><br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/Achievements'><IMG SRC="http://farm4.staticflickr.com/3739/10529350025_2db22731e9.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/Achievements'>Grand Prize Winner, North America<br>Best Experimental Measurement Approach, North America</a></TD><br />
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<TR><TD ALIGN="center"><a href='http://articles.philly.com/2013-11-27/news/44490110_1_synthetic-biology-research-project-international-genetically-engineered-machine'><IMG SRC="http://farm8.staticflickr.com/7397/10529350205_c12e2cd367.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='http://articles.philly.com/2013-11-27/news/44490110_1_synthetic-biology-research-project-international-genetically-engineered-machine'>Featured in a Philadelphia Inquirer article</a> <br> <br> <br></TD><br />
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</html></div>Tyckohttp://2013.igem.org/Team:PennTeam:Penn2014-01-15T16:42:02Z<p>Tycko: </p>
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<br><a href = 'https://docs.google.com/forms/d/188iefj_j6gBvrHC0IS3rG2zBelaVuy-JddYNN7QF8_Y/viewform'>Interested in joining the team? Submit your application for Penn iGEM 2014 here. </a><br><br />
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<center><object width="900" height="506"><param name="movie" value="//www.youtube.com/v/YJQniNpnU9Q?version=3&amp;hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="//www.youtube.com/v/YJQniNpnU9Q?version=3&amp;hl=en_US" type="application/x-shockwave-flash" width="800" height="506" allowscriptaccess="always" allowfullscreen="true"></embed></object></center><br />
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</TABLE><br><a href='https://2013.igem.org/Team:Penn/MaGellinToolbox'>Constructed a toolbox for developing site-specific DNA methylases</a></TD><br />
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<TD ALIGN = "center" width='240'> <TABLE <br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/AssayOverview'><IMG SRC="http://farm3.staticflickr.com/2836/10529350575_dfb38e3538.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/AssayOverview'>Created a standardized assay for site-specific methylases</a></TD><br />
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<TD ALIGN = "center" width='240'> <TABLE <br />
style="border:10px inset #2D68B6;"><br />
<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/Software'><IMG SRC="http://farm4.staticflickr.com/3742/10529401844_cc5f713a48.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/Software'>Developed software to automate analysis of our assay</a></TD><br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/MethylaseOverview'><IMG SRC="http://farm6.staticflickr.com/5478/10529350195_2c54c496e5.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/MethylaseOverview'>Designed and characterized a novel TALE-methylase fusion protein</a> <br></TD><br />
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<TD ALIGN = "center" width='240'> <TABLE <br />
style="border:10px inset #F61F27;"><br />
<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/hpoverview'><IMG SRC="http://farm4.staticflickr.com/3670/10529586493_833c6216ee.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/hpoverview'>Introduced synthetic biology to the community</a></TD><br />
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<TD ALIGN = "center" width='240'> <TABLE <br />
style="border:10px inset #ED115B;"><br />
<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/Too_Soon_To_Treat'><IMG SRC="http://farm4.staticflickr.com/3815/10529365086_82d164e2b1.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/Too_Soon_To_Treat'>Published an article on the ethics of epigenetic engineering</a></TD><br />
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</TABLE><br><a href='https://2013.igem.org/Team:Penn/Team'>Our team</a> <br> <br> <br></TD><br />
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</TABLE><br><a href='https://2013.igem.org/Team:Penn/Achievements'>Grand Prize Winner, North America<br>Best Experimental Measurement Approach, North America</a></TD><br />
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</TABLE><br><a href='http://articles.philly.com/2013-11-27/news/44490110_1_synthetic-biology-research-project-international-genetically-engineered-machine'>Featured in a Philadelphia Inquirer article</a> <br> <br> <br></TD><br />
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<header><h1><b><center>Achievements</b></h1></header><br />
<p><!--write accomplishments here--><br />
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<br/><h7><br />
Our goal was to enable epigenetic engineering. We believe synthetic biological systems should be as subtle and robust as their naturally occurring counterparts. DNA methylation has potential as an orthogonal means of silencing bacterial transcription, irrespective of the promoter.</h7><br/><br />
<ol><br />
<li><a href="https://2013.igem.org/Team:Penn/MaGellinMotivation">Validated a novel methylation assay, MaGellin, to accelerate development of epigenetic engineering tools.</a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/FusionMotivation">Designed and characterized a functional fusion protein to facilitate epigenetic engineering. De-noised the complex TAL-effector system, with implications for other genome engineering applications.</a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/MaGellinSoftware">Built a complementary MaGellin software package to automate experimental analysis. Uniquely, it can interpret methylation-sensitive DNA digestion and then interpret gel electrophoresis images. It is being developed for use by the DIYBio community.</a></li><br />
<li><a href="https://dl.dropboxusercontent.com/u/11828463/MaGellin%20Spec%20Sheet.pdf">Created a specification sheet for the MaGellin Assay workflow, to facilitate quick development of site-specific methylases.</a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/Biobricks">BioBrick'ed our MaGellin plasmid so other iGEM teams can build off our work</a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/Biobricks">BioBrick'ed the M.sssI methylase and the M.sssI methylase with the linker we used for other teams working with methylases.</a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/Too_Soon_To_Treat">Wrote an article on the ethical implications of epigenetic interventions, accepted for publication in the "nation's premier peer-reviewed undergraduate bioethics journal". </a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/LIMS">Developed an open-source Laboratory Information Management System to help teams keep their labs organized. </a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/Outreach">Promoted iGEM, synthetic biology, and our research at numerous outreach events to inspire future scientists</a></li><br />
<li><a href="http://mackinstitute.wharton.upenn.edu/2013/its-not-all-in-the-dna-penn-team-benefits-from-mack-institute-sponsorship-in-synthetic-biology-competition/">Featured in an interview.</a></li><br />
<li><a href="https://2013.igem.org/Jamborees">North American regional Grand Prize winners and winners of Best BioBrick Measurement Approach award for our gel-electrophoresis assay.</a></li><br />
<li><a href="https://twitter.com/NEBiolabs/statuses/390858803836502016">Invited to tour and speak with epigenetics experts at New England Biolabs about our assay and project.</a></li><br />
<li><a href="http://mackinstitute.wharton.upenn.edu/2013/2013-penn-igem-team-on-their-regional-championship-victory/">Published on the Mack Institute for Innovation Management website for our regional success.</a></li><br />
<li><a href="http://articles.philly.com/2013-11-27/news/44490110_1_synthetic-biology-research-project-international-genetically-engineered-machine">Featured interviews in the Philadelphia Inquirer.</a></li><br />
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/c/c1/Gelliinn.jpg" alt="InVitro" ><figcaption><i>Danny uses MaGellin to screen a freshly cloned site-specific methylase before the World Jamboree</i></figcaption></figure></div> <br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/MaGellinToolbox'><IMG SRC="http://farm6.staticflickr.com/5523/10529365546_355d2fe7fc.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/MaGellinToolbox'>Constructed a toolbox for developing site-specific DNA methylases</a></TD><br />
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<TD ALIGN = "center" width='240'> <TABLE <br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/AssayOverview'><IMG SRC="http://farm3.staticflickr.com/2836/10529350575_dfb38e3538.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/AssayOverview'>Created a standardized assay for site-specific methylases</a></TD><br />
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<TD ALIGN = "center" width='240'> <TABLE <br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/Software'><IMG SRC="http://farm4.staticflickr.com/3742/10529401844_cc5f713a48.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/Software'>Developed software to automate analysis of our assay</a></TD><br />
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<TD ALIGN = "center" width='240'> <TABLE <br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/MethylaseOverview'><IMG SRC="http://farm6.staticflickr.com/5478/10529350195_2c54c496e5.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/MethylaseOverview'>Designed and characterized a novel TALE-methylase fusion protein</a> <br></TD><br />
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<TD ALIGN = "center" width='240'> <TABLE <br />
style="border:10px inset #F61F27;"><br />
<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/hpoverview'><IMG SRC="http://farm4.staticflickr.com/3670/10529586493_833c6216ee.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/hpoverview'>Introduced synthetic biology to the community</a></TD><br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/Too_Soon_To_Treat'><IMG SRC="http://farm4.staticflickr.com/3815/10529365086_82d164e2b1.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/Too_Soon_To_Treat'>Published an article on the ethics of epigenetic engineering</a></TD><br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/Team'><IMG SRC="http://farm6.staticflickr.com/5533/10529401504_677c4f296a.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/Team'>Our team</a> <br> <br> <br></TD><br />
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<TR><TD ALIGN="center"><a href='https://2013.igem.org/Team:Penn/Achievements'><IMG SRC="http://farm4.staticflickr.com/3739/10529350025_2db22731e9.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='https://2013.igem.org/Team:Penn/Achievements'>Grand Prize Winner, North America<br>Best Experimental Measurement Approach, North America</a></TD><br />
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<TD ALIGN = "center" width='240'> <TABLE <br />
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<TR><TD ALIGN="center"><a href='http://articles.philly.com/2013-11-27/news/44490110_1_synthetic-biology-research-project-international-genetically-engineered-machine'><IMG SRC="http://farm8.staticflickr.com/7397/10529350205_c12e2cd367.jpg" width="240" height="auto"></a></TD></TR><br />
</TABLE><br><a href='http://articles.philly.com/2013-11-27/news/44490110_1_synthetic-biology-research-project-international-genetically-engineered-machine'>Featured in a Philadelphia Inquirer article</a> <br> <br> <br></TD><br />
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<h1><b><center>About the Team</center></b></h1><br />
<div align="left">The Penn iGEM 2013 team is made up of 5 undergraduate students and various advisors and mentors at the University of Pennsylvania. The team comes from various academic disciplines such as math, bioengineering, and computer science. The team works out of the undergraduate bioengineering laboratory and is rumored to live there. <br />
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<h3>The Team Members</h3> <!--title of section--><br />
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<h2>Brad "Big Dark" Kaptur</h2><p>Bioengineering</p><br />
<p>"Screw minipreps, gotta get big."</p> <br />
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<h2>Josh Tycko</h2><p>Mathematics and Biology</p><br />
<p> "Don't worry, I know a guy."</p> <br />
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<h2>Danielle Fields</h2><p>Bioengineering</p><br />
<p>"I have a Starbucks Gold Card. Literally."</p> <br />
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<h2>Danny Cabrera</h2><p>Computational Biology</p><br />
<p>"Yo, [mumble] you pour [mumble] that gel yet?"</p> <br />
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<div class="member_divide"><h2>Mahamad "Dusty" Charawi</h2><p>Management and Computer Science</p><br />
<p>"Hi my name Mahamad, from Penn iGEM."</p> <br />
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<div class="member_divide_text"><h2><a href='http://www.seas.upenn.edu/directory/profile.php?ID=178'>Brian Chow, Ph.D</a></h2><p>Assistant Professor</p><br />
<p>Chow's laboratory aims to create technological innovations to enhance therapeutic interventions in CNS disorders, and to this end, focuses on engineering tools to elucidate, diagnose and treat the diseased brain. He was instrumental in the direction of the team, teaching us science, organization, and leadership.</p> <br />
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<div class="member_divide_text"><h2>Spencer "STG" Glantz</h2><p>Bioengineering (PhD Student)</p><br />
<p>Spencer helped found the first iGEM team at the University of Pennsylvania and has been lending us his deep knowledge and experience with synthetic biology ever since. He helped us every day and truly made this project possible.</p> <br />
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<div class="member_divide_text"><h2><a href='http://www.orkantelhan.com/'>Orkan Telhan, Ph.D</a></h2><p>Assistant Professor</p><br />
<p> Interdisciplinary artist, designer, and researcher whose investigations focus on the design of interrogative objects, interfaces, and media, engaging with critical issues in social, cultural, and environmental responsibility. He greatly assisted the team with our efforts to properly communicate synthetic biology to the public.</p> <br />
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<div class="member_divide_text"><h2><a href='http://bioengineering.rice.edu/Content.aspx?id=4294967626'>Jordan Miller, Ph.D</a></h2><p>Assistant Professor of Bioengineering at Rice</p><br />
<p>Jordan has been an advisor to Penn iGEM teams for the last three years. He has given us insightful scientific advice and words of wisdom, and he sends the best emails.</p> <br />
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<div class="member_divide_text"><h2>Avin "Shiii" Veerakumar </h2><p>iGEM Guru</p><br />
<p>The Penn iGEM Godfather. He sent us great GIFs and drew a computer.</p> <br />
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<div class="member_divide_text"><h2>Michael Salvatore "Gumbaicci Margaricci" Magaraci</h2><p>iGEM Guru</p><br />
<p>Cloning Master. Diagram Master. He was a huge help.</p> <br />
</div><br />
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<!--bubble that says "advisors"--><br />
<h3>Nothing would be possible without these guys</h3> <!--title of section--><br />
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<div class="member_divide_text"><h2>Sevile Mannickarottu</h2><p>Director of Bioengineering Instructional Laboratories</p><br />
<p>The 2013 Penn iGEM team would like to thank Sevile for providing his facilities and equipment for use during the project.</p> <br />
</div><br />
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<div class="member_divide_text"><h2>Henry "HMFM" Ma </h2><p>Bioengineering (Masters Student)</p><br />
<br />
<p>"Coffee?"</p> <br />
<p>Thank you to Henry for his wonderful support throughout this whole process.</p><br />
</div><br />
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<br><br />
<p style="text-align: center"><b>We could not have succeeded without the help of all these incredibly talented people. We are extremely grateful for their help.</b></p><br />
<p style="text-align: center">Generously donated interesting organisms and scientific expertise by <a href='http://www.bio.upenn.edu/people/mark-goulian'>Dr. Mark Goulian</a></p><br />
<p style="text-align: center">Advice on epigenetics by Drs <a href='http://www.med.upenn.edu/apps/faculty/index.php/g20000320/p13534'>Marisa Bartolomei</a> and <a href='http://www.med.upenn.edu/apps/faculty/index.php/g361/p11818'>Rebecca Simmons</a></p><br />
<p style="text-align: center">Advice on methylation assays by Chris Krapp</p><br />
<p style="text-align: center">Computer Vision algorithm by Eric Kauderer Abrams</p><br />
<p style="text-align: center">Graphic Design by Micah Kaats</p><br />
<p style="text-align: center">Assistance developing MaGellin user interface by Morgan Snyder</p><br />
<p style="text-align: center">Video editing and Animation by Dylan Petro</p><br />
<p style="text-align: center">Cinematography and Photography by Jake Whritner</p><br />
<p style="text-align: center">Music by Ben Brodie</p><br />
<p style="text-align: center">Photography by Erica Sacshe</p><br />
<p style="text-align: center">Web development by Dylan Petro and Stefanie Alfonso</p><br />
<p style="text-align: center">We would also like to thank <a href='http://davidgdula.com'>David Gdula</a> of NEB for being so personally invested in the team's success</p><br />
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<header><h1><b><center>The MaGellin Software Package</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
</br><br />
<h4><b>Overview</b></h4> It was clear that not all gels would be simple or fast to read by eye, and we wanted to be able to quantify relative intensities of bands between samples. We also wanted to solve our unique problem of calculating how band lengths will change when there’s full methylation, site-specific methylation, or no methylation. The solution can change whenever you clone in a new site-specific methylase. We took this problem to the world’s largest college hackathon (PennApps), and had a functional software package after a <a href="http://www.redbull.com/us/en">sleepless</a> 48 hours. It has since been further refined, and certain elements will be made available to the DIYBio community alongside standardized hardware through collaboration with the biotech start-up <a href="http://genefoo.com/">GeneFoo.</a><br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/0/0b/Penn1.png" alt="MaGellinWorkflow1" width="600" ><figcaption><i>The MaGellin Software Package is part of the MaGellin Assay workflow, automating complex data analysis.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
The MaGellin Software Package is a MATLAB script that uses computer vision algorithms to calculate the location and intensity of DNA bands. It also has a bioinformatics module to compare the lengths of bands with the expected lengths, based on the methylation sensitivity of the enzymes and the sequence of the plasmid. So, this program is more than another gel quantifier - it interprets the biological meaning of the band lengths and returns experimentally relevant analyses. The MaGellin Software Package is crucial for our workflow because it allows for clear input/output. Since our restriction digests always yield bands in predictable locations, user bias is eliminated and data sets are standardized across trials and labs, accelerating the pace of discovery. <br />
</br><br />
</br><br />
</br><br />
<b>Protocol</b><br />
<ol><br />
<li>Upload gel picture from restriction enzyme digest. Fill out all relevant information on the graphical user interface. Enter plasmid and target sequences and select restriction enzymes used. Enter descriptive names for gel and lanes if desired. </li><br />
<li>Press the Analyze button.</li> <br />
<li>The Magellin Software Package will calculate the intensity and position of each band and produce a graph. Save your results. </li><br />
<li>Collaborate with your fellow scientists by sharing your results on SkyDrive.</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/b/bc/Penn2.png" alt="MaGellinWorkflow2" width="600" ><figcaption><i>The Magellin Software Package will calculate the intensity and position of each band and produce a graph.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Details on the Software's Inner Workings.</b></h4> Before the analysis is performed, the image is subject to various procedures intended to remove ambient noise in the data. These methods include determining the average amount of noise bias per row, and convolving the image with several standard filters. Next, the MaGellin software finds the lanes in the gel. This is accomplished by running an edge detection algorithm that uses the location of edges of individual bands to infer the location of the center of each lane. The edge detection algorithm functions by treating edges as solutions to variational equations. Once the center of each lane is determined, MaGellin Software is ready to perform its analysis. The user inputs the plasmid sequence in question, and MaGellin Software converts these values into vertical coordinates on the gel by performing a logarithmic regression on the length of the user input sequences, relying on the factory-included values for the lengths of the bands of the NEB 2-Log ladder. Now MaGellin knows where to look, and it knows what it is looking for. All that’s left to do is run the analysis. MaGellin searches in the locations in question, and if a band of suitably high intensity is found, Magellin records the intensity at this location. If no such band is found, Magellin records the intensity as 0 to indicate that no band was found in the area. The data is then extrapolated into a graph with the relevant biological meaning. Usually, we perform our experiments in triplicate, so we can run a 2-way ANOVA. <br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/5/55/Penn3.png" alt="MaGellinWorkflow3" width="700" ><figcaption><i>MaGellin uses a complex edge detection and visualization algorithm to compute band intensities and quanify gel data.</i></figcaption></figure></div><br />
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<header><h1><b><center>The MaGellin Software Package</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
</br><br />
<h4><b>Overview</b></h4> It was clear that not all gels would be simple or fast to read by eye, and we wanted to be able to quantify relative intensities of bands between samples. We also wanted to solve our unique problem of calculating how band lengths will change when there’s full methylation, site-specific methylation, or no methylation. The solution can change whenever you clone in a new site-specific methylase. We took this problem to the world’s largest college hackathon (PennApps), and had a functional software package after a sleepless 48 hours. It has since been further refined, and certain elements will be made available to the DIYBio community alongside standardized hardware through collaboration with the biotech start-up <a href="http://genefoo.com/">GeneFoo.</a><br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/0/0b/Penn1.png" alt="MaGellinWorkflow1" width="600" ><figcaption><i>The MaGellin Software Package is part of the MaGellin Assay workflow, automating complex data analysis.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
The MaGellin Software Package is a MATLAB script that uses computer vision algorithms to calculate the location and intensity of DNA bands. It also has a bioinformatics module to compare the lengths of bands with the expected lengths, based on the methylation sensitivity of the enzymes and the sequence of the plasmid. So, this program is more than another gel quantifier - it interprets the biological meaning of the band lengths and returns experimentally relevant analyses. The MaGellin Software Package is crucial for our workflow because it allows for clear input/output. Since our restriction digests always yield bands in predictable locations, user bias is eliminated and data sets are standardized across trials and labs, accelerating the pace of discovery. <br />
</br><br />
</br><br />
</br><br />
<b>Protocol</b><br />
<ol><br />
<li>Upload gel picture from restriction enzyme digest. Fill out all relevant information on the graphical user interface. Enter plasmid and target sequences and select restriction enzymes used. Enter descriptive names for gel and lanes if desired. </li><br />
<li>Press the Analyze button.</li> <br />
<li>The Magellin Software Package will calculate the intensity and position of each band and produce a graph. Save your results. </li><br />
<li>Collaborate with your fellow scientists by sharing your results on SkyDrive.</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/b/bc/Penn2.png" alt="MaGellinWorkflow2" width="600" ><figcaption><i>The Magellin Software Package will calculate the intensity and position of each band and produce a graph.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Details on the Software's Inner Workings.</b></h4> Before the analysis is performed, the image is subject to various procedures intended to remove ambient noise in the data. These methods include determining the average amount of noise bias per row, and convolving the image with several standard filters. Next, the MaGellin software finds the lanes in the gel. This is accomplished by running an edge detection algorithm that uses the location of edges of individual bands to infer the location of the center of each lane. The edge detection algorithm functions by treating edges as solutions to variational equations. Once the center of each lane is determined, MaGellin Software is ready to perform its analysis. The user inputs the plasmid sequence in question, and MaGellin Software converts these values into vertical coordinates on the gel by performing a logarithmic regression on the length of the user input sequences, relying on the factory-included values for the lengths of the bands of the NEB 2-Log ladder. Now MaGellin knows where to look, and it knows what it is looking for. All that’s left to do is run the analysis. MaGellin searches in the locations in question, and if a band of suitably high intensity is found, Magellin records the intensity at this location. If no such band is found, Magellin records the intensity as 0 to indicate that no band was found in the area. The data is then extrapolated into a graph with the relevant biological meaning. Usually, we perform our experiments in triplicate, so we can run a 2-way ANOVA. <br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/5/55/Penn3.png" alt="MaGellinWorkflow3" width="700" ><figcaption><i>MaGellin uses a complex edge detection and visualization algorithm to compute band intensities and quanify gel data.</i></figcaption></figure></div><br />
</br><br />
</br><br />
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<b><center><h1><br />
PennApps<br />
</b></center></h1><br />
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<br />
Synthetic Biology holds its roots in electrical engineering and computer science, however, most engineers in these fields have had little exposure to Synthetic Biology. To reach out to hundreds of fellow computer scientists and build an open source methylation analysis software along the way, the members of Penn iGEM participated in PennApps, the largest college hackathon. PennApps is attended by approximately 1,000 top student hackers from around the world. Over the course of 48 hours, teams work to design an app from start to finish. At the demo round, all the teams – over 200 in total – present their apps to fellow hackers, interested students, and representatives from the tech industry. The Penn iGEM team represented synthetic biology at an event predominantly attended by computer science students, and PennApps attendees were excited to see a new computer program with a scientific application.<br />
<br />
<br />
<br/><br />
<br/><br />
<br><br />
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<p>Our booth is busy! Danny and Josh simultaneously present the<br> app to two interested onlookers.</p><br />
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<div style="float:right;"> <br />
<img src="https://static.igem.org/mediawiki/2013/b/ba/Penn4.jpg" width='420' height='auto'><br />
<p>Josh explains the math behind the software.</p><br />
</div><br />
<div style="clear:both"/><br />
<br/><br />
<br/><br />
Our final product was a gel analysis app which automates the process of interpreting DNA bands run through gel electrophoresis. This app, the Magellin Software Package, complements our digestion assay and reports the extent of on-target and off-target methylation, greatly simplifying data analysis for the experimenter. Unlike other gel analysis programs, MaGellin interprets plasmid sequences, understands methylation patterns, and assesses targeted methylation based on a gel image.<br />
<br><br />
<br><br />
Learn more about the MaGellin Software Package on our <a href="https://2013.igem.org/Team:Penn/Software">software</a> page.<br />
<br><br />
<br><br />
The MaGellin Software Package is a robust platform that can easily be adjusted for other gel analysis problems, such as interpreting COBRA and quantifying PCR yields. We would like to collaborate with other iGEM teams to expand the range of gel analysis tasks this software performs.<br />
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<b><center><h1><br />
Project Overview<br />
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<p><br />
The code of life is more than a sequence of A’s, C’s, T’s, and G’s. Heart cells in the human body contain the same DNA as skin cells in the foot, yet these two cell types behave in radically different ways. Both contain the DNA for every one of over 20,000 human genes but express only the ones needed for their own form and function. These differences are due to epigenetic controls. Epigenetics refers to any regulation of gene expression and phenotype that is not based on the sequence of bases in DNA. In addition to governing cellular differentiation, epigenetic mechanisms facilitate the proper functioning of a cell. When these mechanisms go awry, neurodevelopmental disorders, immunodeficiency, and cancer can result. Epigenetic phenomena are amongst the primary ways gene expression is regulated; yet, our current understanding of them is limited, especially due to the challenge of studying them in noisy mammalian systems. By virtue of its emphasis on the isolation and testing of biological networks in engineered systems with reduced complexity, synthetic biology offers the promise of fostering understanding of phenomena difficult to study in their native environments. It is thus an ideal forum for epigenetic studies. At its core, synthetic biology also involves the engineering of non-native networks for useful purposes. Well-tuned gene expression is essential to the proper functioning of synthetic biological circuitry, yet epigenetics has not yet been fully explored as a tool for this application. <br />
<br />
</br></br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/4/4b/Penn_Toolbox.png" alt="Toolbox" width="700" height="395"><figcaption><i>There are three major components to our toolbox:<a href="https://2013.igem.org/Team:Penn/AssayOverview"> the assay</a>,<a href="https://2013.igem.org/Team:Penn/Software"> the software</a>, and the <a href="https://2013.igem.org/Team:Penn/MethylaseOverview">fusion protein</a></i></figcaption></figure></div><br />
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<header><h1><b><center>Achievements</b></h1></header><br />
<p><!--write accomplishments here--><br />
<br />
<br/><h7><br />
Our goal was to enable epigenetic engineering. We believe synthetic biological systems should be as subtle and robust as their naturally occurring counterparts. DNA methylation has potential as an orthogonal means of silencing bacterial transcription, irrespective of the promoter.</h7><br/><br />
<ol><br />
<li><a href="https://2013.igem.org/Team:Penn/MaGellinMotivation">Validated a novel methylation assay, MaGellin, to accelerate development of epigenetic engineering tools.</a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/FusionMotivation">Designed and characterized a functional fusion protein to facilitate epigenetic engineering. De-noised the complex TAL-effector system, with implications for other genome engineering applications.</a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/MaGellinSoftware">Built a complementary MaGellin software package to automate experimental analysis. Uniquely, it can interpret methylation-sensitive DNA digestion and then interpret gel electrophoresis images. It is being developed for use by the DIYBio community.</a></li><br />
<li><a href="https://dl.dropboxusercontent.com/u/11828463/MaGellin%20Spec%20Sheet.pdf">Created a specification sheet for the MaGellin Assay workflow, to facilitate quick development of site-specific methylases.</a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/Biobricks">BioBrick'ed our MaGellin plasmid so other iGEM teams can build off our work</a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/Biobricks">BioBrick'ed the M.sssI methylase and the M.sssI methylase with the linker we used for other teams working with methylases.</a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/Too_Soon_To_Treat">Wrote an article on the ethical implications of epigenetic interventions, accepted for publication in the "nation's premier peer-reviewed undergraduate bioethics journal". </a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/LIMS">Developed an open-source Laboratory Information Management System to help teams keep their labs organized. </a></li><br />
<li><a href="https://2013.igem.org/Team:Penn/Outreach">Promoted iGEM, synthetic biology, and our research at numerous outreach events to inspire future scientists</a></li><br />
<li><a href="http://mackinstitute.wharton.upenn.edu/2013/its-not-all-in-the-dna-penn-team-benefits-from-mack-institute-sponsorship-in-synthetic-biology-competition/">Featured in an interview.</a></li><br />
<li><a href="https://2013.igem.org/Jamborees">North American regional Grand Prize winners and winners of Best BioBrick Measurement Approach award for our gel-electrophoresis assay.</a></li><br />
<li><a href="https://twitter.com/NEBiolabs/statuses/390858803836502016">Invited to tour and speak with epigenetics experts at New England Biolabs about our assay and project.</a></li><br />
<li><a href="http://mackinstitute.wharton.upenn.edu/2013/2013-penn-igem-team-on-their-regional-championship-victory/">Published on the Mack Institute for Innovation Management website for our regional success.</a></li><br />
</ol><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/c/c1/Gelliinn.jpg" alt="InVitro" ><figcaption><i>Danny uses MaGellin to screen a freshly cloned site-specific methylase before the World Jamboree</i></figcaption></figure></div> <br />
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</TABLE><br><a href='https://2013.igem.org/Team:Penn/MaGellinToolbox'>Constructed a toolbox for developing site-specific DNA methylases</a></TD><br />
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</TABLE><br><a href='https://2013.igem.org/Team:Penn/MethylaseOverview'>Designed and characterized a novel TALE-methylase fusion protein</a> <br></TD><br />
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</TABLE><br><a href='https://2013.igem.org/Team:Penn/Too_Soon_To_Treat'>Published an article on the ethics of epigenetic engineering</a></TD><br />
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</TABLE><br><a href='https://2013.igem.org/Team:Penn/Achievements'>Grand Prize Winner, North America<br>Best Experimental Measurement Approach, North America</a></TD><br />
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</TABLE><br><a href='https://2013.igem.org/Team:Penn/MethylaseOverview'>Designed and characterized a novel TALE-methylase fusion protein</a><br> <br></TD><br />
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</TABLE><br><a href='https://2013.igem.org/Team:Penn/Too_Soon_To_Treat'>Published an article on the ethics of epigenetic engineering</a></TD><br />
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<header><h1><b><center>Site-Specific Methylases</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
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After validating the MaGellin assay with non-specific methylases, we were ready to test site-specific methylases. Our MaGellin assay is ideal for high-throughput construction and testing of these enzymes. Site-specific methylases are fusion proteins: a DNA binding domain linked to a methylase by a serine glycine chain. They can direct DNA methylation to specific sequences, likely promoter regions for use as a transcriptional silencer. We used the prokaryotic methylase M.SssI for all of our studies (BBa_K1128000). <br />
<p>We wanted to first recapitulate published results with a zinc finger binding domain, and then characterize a novel site-specific methylase using a TALE DNA binding domain.<br />
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<header><h1><b><center>Site-Specific Methylases</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
After validating the MaGellin assay with non-specific methylases, we were ready to test site-specific methylases. Our MaGellin assay is ideal for high-throughput construction and testing of these enzymes. Site-specific methylases are fusion proteins: a DNA binding domain linked to a methylase by a serine glycine chain. They can direct DNA methylation to specific sequences, likely promoter regions for use as a transcriptional silencer. We used the prokaryotic methylase M.SssI for all of our studies (BBa_K1128000). <br />
<p>We wanted to first recapitulate published results with a zinc finger binding domain, and then characterize a novel site-specific methylase using a TALE DNA binding domain.<br />
</br></br><br />
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<header><h1><b><center>Site-Specific Methylases</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
After validating the MaGellin assay with non-specific methylases, we were ready to test site-specific methylases. Our MaGellin assay is ideal for high-throughput construction and testing of these enzymes. Site-specific methylases are fusion proteins: a DNA binding domain linked to a methylase by a serine glycine chain. They can direct DNA methylation to specific sequences, likely promoter regions for use as a transcriptional silencer. We used the prokaryotic methylase M.SssI for all of our studies (BBa_K1128000). <br />
<p>We wanted to first recapitulate published results with a zinc finger binding domain, and then characterize a novel site-specific methylase using a TALE DNA binding domain.<br />
</br></br><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:</br><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.</br><br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.</br><br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.</h7><br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified (Xu et al., 1997).<h7> MaGellin agreed with previously published work</h7>, from various groups with no common, standardized assay. It's possible MaGellin is more sensitive to off target methylation than their assays because <h7>MaGellin only reports site-specific methylation if 100% of the methylations is site-specific for that plasmid.</h7><br />
</br><br />
</br><h4><center>ZF-M.SssI fully methylates MaGellin plasmid</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/6/60/Zf-102813.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: A ZF-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 (N) control has no T7 polymerase and no possibility of leaky expression. The linearized control (L) is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. MaGellin reported off target activity, irrespective of zinc finger binding (Figure 1). <h7>MaGellin is sensitive to off target effects</h7> because there is no background CpG methylation in E.coli and the plasmid is short compared to a mammalian genome. We are interested in examining this initial finding further to determine its reproducibility and relevance to previously published zinc finger-methylases.<br />
</br><br />
<br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct (Cong et al., 2012). They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI partially methylates on and off target sites on MaGellin plasmid</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. The off target methylation was significantly reduced compared to what MaGellin reported for the zinc finger fusion. <h7>This showed MaGellin can detect changes in the site-specificity of methylation due to swapping DNA binding domains</h7>. We still expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). <h7>This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty (Xiong et al., 1997).</h7><br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, <h7>it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its fuction <i>in vivo</i></h7>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis (less than 5% the cost of bisulfite sequencing).<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing data for samples marked *. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<br />
We varied induction conditions, expecting one might be more optimal than our initial inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase was still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7><br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
<h7>MaGellin was designed to optimize the development of robust tools for site-specific methylation.</h7> To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<p></br>MaGellin agreed with previously published work that zinc finger methylases are prone to off target methylation. <h7>We were able to construct and test this construct in the three weeks after regionals because of MaGellin's simple workflow.</h7><br />
<br />
<p></br><h7>We picked up on the noisiness of our TALE-M.SssI using MaGellin,</h7> which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal.<h7> Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites,</h7> along the lines of how TALE-Nucleases cleave DNA (Li et al, 2007). <br />
</br></br><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:</br><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.</br><br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.</br><br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.</h7><br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified (Xu et al., 1997).<h7> MaGellin agreed with previously published work</h7>, from various groups with no common, standardized assay. It's possible MaGellin is more sensitive to off target methylation than their assays because <h7>MaGellin only reports site-specific methylation if 100% of the methylations is site-specific for that plasmid.</h7><br />
</br><br />
</br><h4><center>ZF-M.SssI fully methylates MaGellin plasmid</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/6/60/Zf-102813.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: A ZF-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 (N) control has no T7 polymerase and no possibility of leaky expression. The linearized control (L) is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. MaGellin reported off target activity, irrespective of zinc finger binding (Figure 1). <h7>MaGellin is sensitive to off target effects</h7> because there is no background CpG methylation in E.coli and the plasmid is short compared to a mammalian genome. We are interested in examining this initial finding further to determine its reproducibility and relevance to previously published zinc finger-methylases.<br />
</br><br />
<br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct (Cong et al., 2012). They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI partially methylates on and off target sites on MaGellin plasmid</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. The off target methylation was significantly reduced compared to what MaGellin reported for the zinc finger fusion. <h7>This showed MaGellin can detect changes in the site-specificity of methylation due to swapping DNA binding domains</h7>. We still expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). <h7>This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty (Xiong et al., 1997).</h7><br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, <h7>it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its fuction <i>in vivo</i></h7>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis (less than 5% the cost of bisulfite sequencing).<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing data for samples marked *. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<br />
We varied induction conditions, expecting one might be more optimal than our initial inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase was still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7><br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
<h7>MaGellin was designed to optimize the development of robust tools for site-specific methylation.</h7> To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<p></br>MaGellin agreed with previously published work that zinc finger methylases are prone to off target methylation. <h7>We were able to construct and test this construct in the three weeks after regionals because of MaGellin's simple workflow.</h7><br />
<br />
<p></br><h7>We picked up on the noisiness of our TALE-M.SssI using MaGellin,</h7> which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA (Li et al, 2007). <br />
</br></br><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:</br><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.</br><br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.</br><br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.</h7><br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified (Xu et al., 1997).<h7> MaGellin agreed with previously published work</h7>, from various groups with no common, standardized assay. It's possible MaGellin is more sensitive to off target methylation than their assays because <h7>MaGellin only reports site-specific methylation if 100% of the methylations is site-specific for that plasmid.</h7><br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/6/60/Zf-102813.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: A ZF-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 (N) control has no T7 polymerase and no possibility of leaky expression. The linearized control (L) is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. MaGellin reported off target activity, irrespective of zinc finger binding (Figure 1). <h7>MaGellin is sensitive to off target effects</h7> because there is no background CpG methylation in E.coli and the plasmid is short compared to a mammalian genome. We are interested in examining this initial finding further to determine its reproducibility and relevance to previously published zinc finger-methylases.<br />
</br><br />
<br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct (Cong et al., 2012). They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). <h7>This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty (Xiong et al., 1997).</h7><br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, <h7>it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its fuction <i>in vivo</i></h7>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis (less than 5% the cost of bisulfite sequencing).<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing data for samples marked *. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<br />
We varied induction conditions, expecting one might be more optimal than our initial inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase was still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7><br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
<h7>MaGellin was designed to optimize the development of robust tools for site-specific methylation.</h7> To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<p></br>MaGellin agreed with previously published work that zinc finger methylases are prone to off target methylation. <h7>We were able to construct and test this construct in the three weeks after regionals because of MaGellin's simple workflow.</h7><br />
<br />
<p></br><h7>We picked up on the noisiness of our TALE-M.SssI using MaGellin,</h7> which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA (Li et al, 2007). <br />
</br></br><br />
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<b><center><h1><br />
Future Directions<br />
</b></center></h1><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br></br><br />
</br><br />
<h4><b>Advantages of a TALE-methylase</b></h4><br />
<p>Our new TALE fusion is much more modular and easy to customize than the old zinc finger (Table 1). TALE construction is not heavily patented like zinc-finger design, and TALE’s are considerably cheaper to produce (Sanjana 2012 and Zhang 2011). They have made the zinc-fingers nearly obsolete as a tool for genetic engineering, including the creation of transgenic animal models (Tesson 2011, Sander 2011, Huang 2011, and Zhang 2011). We expect our new fusion protein could be translated to enable the creation of differentially methylated animal models, which would be revolutionary for epigenetic disease research (Klose 2006). In effect, this could transform epigenetics from a largely observational discipline to one of active intervention and manipulation, similar to the transition from early classical genetics to genetic engineering and synthetic biology.<br />
</br><br />
<br />
<h4><b>CRISPR System</b></h4><p>We also fused a methyltransferase to a dCas9 DNA binding domain from the CRISPR-Cas system. We modified our MaGellin vector to include a cassette that expresses the necessary guiding sgRNA1. The sgRNA can easily be swapped out to quickly re-target the Cas system, much more quickly and with less synthesis than it would take to re-target the TALE. We are intrigued by the prospect of optimizing the Cas system, which is considered to possibly have many advantages over the TALE (Gaj 2013). It has already been fused to transcription activators that similarly target promoters, so the strategy is theoretically sound (Bikard 2013). Particularly, it’s ability to multiplex and target multiple sites for methylation would be very useful (Wang 2013). However, many recent papers have described problems with Cas specificity that seriously limit its usefulness, so we expect the excitement around Cas may dim as scientists begin a rigorous re-evaluation of the comparison between Cas and TALE systems. We propose MaGellin as a simple, fast, and inexpensive assay to aid the development of superior Cas systems, as they are optimized by site-directed mutagenesis, directed evolution, or other methods. As outlined above and demonstrated with our TALE, fusing a methylase to the Cas serves as a detectable marker for its DNA binding.</br><br />
<h4><b>Split-Reconstitution Site-Specific Methylases</b></h4><br />
</br>Most previously published zinc finger-methylase studies have used the same two components as our TALE, the DNA binding domain linked to the methylase. In the noiseless MaGellin system, we were able to detect significant off target methylation at long induction times and high IPTG concentrations. This suggests these fusions still methylate in a non-specific manner once the binding sites are saturated. This issue must be harder to detect in mammalian systems. We are proposing the best strategy would be to split the methylase into subunits that only dimerize and activate at the target site when co-localized by their linked TALEs. This has been effective for TALE nucleases.<br />
</br>We are going to try this strategy by using the other multiple cloning site on the MaGellin plasmid, which we have so far used to express an sgRNA for the dCas9-M.SssI.<br />
<br />
<h4><b>Translational Potential</b></h4><br />
<p>It is worth noting that the TALE-methylase can be delivered to mammalian cells by adeno-associated vectors (AAV). This viral vector is the best currently available in terms of safety and efficiency (Daya 2008). Different serotypes have different cell tropisms, which can provide efficient cell-type targeting when used in conjunction with a cell-specific promoter (Ellis 2013). As we consider different model organisms, we may also want to try different methyltransferases. Importantly, the TALE, at 2.5kb long, can easily be packaged with a methyltransferase in an AAV vector, whereas a Cas would not fit (Konermann 2013).<br />
<br />
<h4><b>Epigenome Engineering is a Reality Today</b></h4><br />
<p>We were not the only synthetic biologists developing epigenetic engineering tools this summer. In August, we were excited to see George Church’s lab at Harvard had fused TALEs to histone modifiers, enabling another distinct form of epigenetic engineering (Konermann 2013). They delivered these TALE fusions to mammalian cells with AAV, which gives us confidence our system can be translated into more complex organisms. With their targeted histone modification and our targeted DNA methylation, it appears the era of serious epigenetic engineering efforts is now upon us. <br />
<br />
<h4><b>Transcriptional Silencing in Bacteria with Methylation</b></h4><br />
<p>At the regional jamboree, we were asked how precisely we could use CpG methylation to silence transcription in E.coli, which have no such endogenous mechanism. In response, we have designed an experiment using MeCP2, a DNA binding protein that only binds when its recognition site is methylated. We hope to use this protein to hinder the polymerase. So far, we have cloned and purified the protein, including a variant which was truncated to remove the DNA binding domain, to serve as our negative control.<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/e/e3/Mecp2withlabels.jpg" height="400" alt="SDS"><figcaption><i>A SDS-Page gel shows expression of the MECP2 gene.</i></figcaption></figure></div></br><br />
<div align=left><br />
<h4><b>Alternative to <i>ChIP.</i> </b></h4><br />
<ol><li>MaGellin can also be used to screen the binding specificity of transcription factors (TF) and other DNA binding domains, with methylation serving as an effective reporter for targeted binding. </li><br />
<br />
<li>By fusing a TF to the methyltransferase, its binding with the plasmid becomes easily detected by the associated methylation</li><br />
<br />
<li>In this situation, one would vary the “target site” to see which DNA sequences have the strongest binding to the TF</li><br />
<br />
<li>This technique would be significantly faster, simpler, and cheaper than a ChIP-based method</li><br />
</ol><br />
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<header><h1><b><center>Assay Validation</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
<h4><b>We have designed standardized bisulfite sequencing primers.</b></h4> Bisulfite sequencing is a good next step after restriction digest to further characterize functional site-specific methylases, but it is inherently very difficult to design good primers. People use advanced algorithms for primer design that are still not guaranteed to successfully sequence some sequences. We went through 8 sets of primers, most of which did not show the proper bias to amplify only bisulfite converted DNA. Primer Set 2 was successful and is included with our MaGellin plasmid, much like VF and VR are included as standardized biobrick sequencing primers (Figure 1).<br />
</br><br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/4/4a/Biseq.png/631px-Biseq.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: Validating standardized bisulfite sequencing primers. Primer Set 2 successfully amplifies bisulfite converted DNA but not unconverted DNA, as desired.</i></figcaption></figure></div><br />
</br></br><br />
<br />
<h4><b>MaGellin effectively detects methylation in vitro.</b></h4> First, we tested MaGellin with a purified methylase in vitro. The results made it clear that MaGellin can detect methylation at both the “target” and “off-target” site (Figure 2). MaGellin is also sensitive to various degrees of methylation (Figure 3). These experiments helped us optimize the ideal amount of plasmid and restriction enzyme to use in any study moving forward.<br />
</br><br />
</br><br />
</br><br />
<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/0/00/InVitroConfirmation2.png" alt="InVitro" width="600" height="395"><figcaption><i>Figure 2: Plasmid DNA treated in vitro with purified M.SssI. The first three lanes were not treated and show zero methylation detection by our assay. The last three lanes were methylated and show 100% methylation. This figure validates that MaGellin is capable of clear input/output.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/5/58/Methylation_timecourse.png" alt="Timecourse" height="395"><figcaption><i>Figure 3: Plasmid DNA treated in vitro with purified M.SssI. Each lane was treated for a different amount of time, this figure shows that MaGellin, alongside our <a href = "https://2013.igem.org/Team:Penn/Software">software package</a>, can report relative differences in the level of methylation.</i></figcaption></figure></div><br />
<br />
</br><br />
</br><br />
</br><br />
<h4><b>MaGellin detects methylation in vivo.</b> </h4> We expressed M.SssI in vivo and compared it with purified M.SssI used on the plasmid in vitro. In both cases, we saw similar full methylation of the plasmid, confirming that MaGellin can express methylases and report their activity in vivo (Figure 4).<br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/3/34/Vivo_validation.png" alt="InVivo" width="600"><figcaption><i>Figure 4: M.SssI expressed in vivo compared with in vitro methylation.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<br />
<br />
<center><h1>Summary</center><br />
<ol><br />
We have created MaGellin, a new technology that facilitates screening novel DNA binding domain – methylase fusion proteins<br />
<li>Our assay is less expensive and faster than existing methods</li><br />
<li>We have eliminated noise associated with previous studies</li><br />
<li>We have a system with clear input/output</li><br />
<li>Our assay lends itself to high throughput screening of many different proteins</li><br />
<li>We are releasing it alongside an open source data analysis software package which streamlines the entire screening process</li><br />
</ol><br />
<br><br />
<br><br />
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<header><h1><b><center>Assay Validation</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
<h4><b>We have designed standardized bisulfite sequencing primers.</b></h4> Bisulfite sequencing is a good next step after restriction digest to further characterize functional site-specific methylases, but it is inherently very difficult to design good primers. People use advanced algorithms for primer design that are still not guaranteed to successfully sequence some sequences. We went through 8 sets of primers, most of which did not show the proper bias to amplify only bisulfite converted DNA. Primer Set 2 was successful and is included with our MaGellin plasmid, much like VF and VR are included as standardized biobrick sequencing primers (Figure 1).<br />
</br><br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/4/4a/Biseq.png/631px-Biseq.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: Validating standardized bisulfite sequencing primers. Primer Set 2 successfully amplifies bisulfite converted DNA but not unconverted DNA, as desired.</i></figcaption></figure></div><br />
</br></br><br />
<br />
<h4><b>MaGellin effectively detects methylation in vitro.</b></h4> First, we tested MaGellin with a purified methylase in vitro. The results made it clear that MaGellin can detect methylation at both the “target” and “off-target” site (Figure 2). MaGellin is also sensitive to various degrees of methylation (Figure 3). These experiments helped us optimize the ideal amount of plasmid and restriction enzyme to use in any study moving forward.<br />
</br><br />
</br><br />
</br><br />
<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/0/00/InVitroConfirmation2.png" alt="InVitro" width="600" height="395"><figcaption><i>Figure 2: Plasmid DNA treated in vitro with purified M.SssI. The first three lanes were not treated and show zero methylation detection by our assay. The last three lanes were methylated and show 100% methylation. This figure validates that MaGellin is capable of clear input/output.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/5/58/Methylation_timecourse.png" alt="Timecourse" height="395"><figcaption><i>Figure 3: Plasmid DNA treated in vitro with purified M.SssI. Each lane was treated for a different amount of time, this figure shows that MaGellin, alongside our software package, can report relative differences in the level of methylation.</i></figcaption></figure></div><br />
<br />
</br><br />
</br><br />
</br><br />
<h4><b>MaGellin detects methylation in vivo.</b> </h4> We expressed M.SssI in vivo and compared it with purified M.SssI used on the plasmid in vitro. In both cases, we saw similar full methylation of the plasmid, confirming that MaGellin can express methylases and report their activity in vivo (Figure 4).<br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/3/34/Vivo_validation.png" alt="InVivo" width="600"><figcaption><i>Figure 4: M.SssI expressed in vivo compared with in vitro methylation.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<br />
<br />
<center><h1>Summary</center><br />
<ol><br />
We have created MaGellin, a new technology that facilitates screening novel DNA binding domain – methylase fusion proteins<br />
<li>Our assay is less expensive and faster than existing methods</li><br />
<li>We have eliminated noise associated with previous studies</li><br />
<li>We have a system with clear input/output</li><br />
<li>Our assay lends itself to high throughput screening of many different proteins</li><br />
<li>We are releasing it alongside an open source data analysis software package which streamlines the entire screening process</li><br />
</ol><br />
<br><br />
<br><br />
<br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:</br><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.</br><br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.</br><br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.</h7><br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified.<h7> MaGellin agreed with previously published work</h7>, from various groups with no common, standardized assay. It's possible MaGellin is more sensitive to off target methylation than their assays because <h7>MaGellin only reports site-specific methylation if 100% of the methylations is site-specific for that plasmid.</h7><br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/6/60/Zf-102813.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: A ZF-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 (N) control has no T7 polymerase and no possibility of leaky expression. The linearized control (L) is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. MaGellin reported off target activity, irrespective of zinc finger binding (Figure 1). <h7>MaGellin is sensitive to off target effects</h7> because there is no background CpG methylation in E.coli and the plasmid is short compared to a mammalian genome. We are interested in examining this initial finding further to determine its reproducibility and relevance to previously published zinc finger-methylases.<br />
</br><br />
<br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). <h7>This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.</h7><br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, <h7>it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its fuction <i>in vivo</i></h7>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis (less than 5% the cost of bisulfite sequencing).<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing data for samples marked *. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<br />
We varied induction conditions, expecting one might be more optimal than our initial inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase was still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7><br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
<h7>MaGellin was designed to optimize the development of robust tools for site-specific methylation.</h7> To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<p></br>MaGellin agreed with previously published work that zinc finger methylases are prone to off target methylation. <h7>We were able to construct and test this construct in the three weeks after regionals because of MaGellin's simple workflow.</h7><br />
<br />
<p></br><h7>We picked up on the noisiness of our TALE-M.SssI using MaGellin,</h7> which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. <br />
</br></br><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:</br><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.</br><br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.</br><br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.</h7><br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified.<h7> MaGellin agreed with previously published work</h7>, from various groups with no common, standardized assay. It's possible MaGellin is more sensitive to off target methylation than their assays because <h7>MaGellin only reports site-specific methylation if 100% of the methylations is site-specific for that plasmid.</h7><br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/6/60/Zf-102813.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: A ZF-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 (N) control has no T7 polymerase and no possibility of leaky expression. The linearized control (L) is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. MaGellin reported off target activity, irrespective of zinc finger binding (Figure 1). <h7>MaGellin is sensitive to off target effects</h7> because there is no background CpG methylation in E.coli and the plasmid is short compared to a mammalian genome. We are interested in examining this initial finding further to determine its reproducibility and relevance to previously published zinc finger-methylases.<br />
</br><br />
<br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). <h7>This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.</h7><br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its fuction <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis (less than 5% the cost of bisulfite sequencing).<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing data for samples marked *. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<br />
We varied induction conditions, expecting one might be more optimal than our initial inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase was still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7><br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
<h7>MaGellin was designed to optimize the development of robust tools for site-specific methylation.</h7> To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<p></br>MaGellin agreed with previously published work that zinc finger methylases are prone to off target methylation. <h7>We were able to construct and test this construct in the three weeks after regionals because of MaGellin's simple workflow.</h7><br />
<br />
<p></br><h7>We picked up on the noisiness of our TALE-M.SssI using MaGellin,</h7> which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. <br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:</br><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.</br><br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.</br><br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.</h7><br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified.<h7> MaGellin agreed with previously published work</h7>, from various groups with no common, standardized assay. It's possible MaGellin is more sensitive to off target methylation than their assays because <h7>MaGellin only reports site-specific methylation if 100% of the methylations is site-specific for that plasmid.</h7><br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/6/60/Zf-102813.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: A ZF-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 (N) control has no T7 polymerase and no possibility of leaky expression. The linearized control (L) is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
</br><i>Figure 1: The zinc finger-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. MaGellin reported off target activity, irrespective of zinc finger binding (Figure 1). <h7>MaGellin is sensitive to off target effects</h7> because there is no background CpG methylation in E.coli and the plasmid is short compared to a mammalian genome. We are interested in examining this initial finding further to determine its reproducibility and relevance to previously published zinc finger-methylases.<br />
</br><br />
<br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). <h7>This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.</h7><br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its fuction <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis (less than 5% the cost of bisulfite sequencing).<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing data for samples marked *. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<br />
We varied induction conditions, expecting one might be more optimal than our initial inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase was still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7><br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
<h7>MaGellin was designed to optimize the development of robust tools for site-specific methylation.</h7> To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<p></br>MaGellin agreed with previously published work that zinc finger methylases are prone to off target methylation. <h7>We were able to construct and test this construct in the three weeks after regionals because of MaGellin's simple workflow.</h7><br />
<br />
<p></br><h7>We picked up on the noisiness of our TALE-M.SssI using MaGellin,</h7> which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. <br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:</br><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.</br><br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.</br><br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.</h7><br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified.<h7> MaGellin agreed with previously published work</h7>, from various groups with no common, standardized assay. It's possible MaGellin is more sensitive to off target methylation than their assays because <h7>MaGellin only reports site-specific methylation if 100% of the methylations is site-specific for that plasmid.</h7><br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/6/60/Zf-102813.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: A ZF-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 (N) control has no T7 polymerase and no possibility of leaky expression. The linearized control (L) is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
</br><i>Figure 1: The zinc finger-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. MaGellin reported off target activity, irrespective of zinc finger binding (Figure 1). <h7>MaGellin is sensitive to off target effects</h7> because there is no background CpG methylation in E.coli and the plasmid is short compared to a mammalian genome. We are interested in examining this initial finding further to determine its reproducibility and relevance to previously published zinc finger-methylases.<br />
</br><br />
<br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). <h7>This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.</h7><br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its fuction <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis (less than 5% the cost of bisulfite sequencing).<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing data for samples marked *. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<br />
We varied induction conditions, expecting one might be more optimal than our initial inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase was still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7><br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. <br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:<br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.</h7><br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified.<h7> MaGellin agreed with previously published work</h7>, from various groups with no common, standardized assay. It's possible MaGellin is more sensitive to off target methylation than their assays because <h7>MaGellin only reports site-specific methylation if 100% of the methylations is site-specific for that plasmid.</h7><br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/6/60/Zf-102813.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: A ZF-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 (N) control has no T7 polymerase and no possibility of leaky expression. The linearized control (L) is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
</br><i>Figure 1: The zinc finger-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. MaGellin reported off target activity, irrespective of zinc finger binding (Figure 1). <h7>MaGellin is sensitive to off target effects</h7> because there is no background CpG methylation in E.coli and the plasmid is short compared to a mammalian genome. We are interested in examining this initial finding further to determine its reproducibility and relevance to previously published zinc finger-methylases.<br />
</br><br />
<br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). <h7>This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.</h7><br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its fuction <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis (less than 5% the cost of bisulfite sequencing).<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing data for samples marked *. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<br />
We varied induction conditions, expecting one might be more optimal than our initial inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase was still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7><br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. <br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:</h7><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.<br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. MaGellin agreed with previously published work, from various groups with no common, standardized assay. It's possible MaGellin is more sensitive to off target methylation than their assays because <h7>MaGellin only reports site-specific methylation if 100% of the methylations is site-specific for that plasmid.</h7><br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/6/60/Zf-102813.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: A ZF-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 (N) control has no T7 polymerase and no possibility of leaky expression. The linearized control (L) is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
</br><i>Figure 1: The zinc finger-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. MaGellin reported off target activity, irrespective of zinc finger binding (Figure 1). <h7>MaGellin is sensitive to off target effects</h7> because there is no background CpG methylation in E.coli and the plasmid is short compared to a mammalian genome. We are interested in examining this initial finding further to determine its reproducibility and relevance to previously published zinc finger-methylases.<br />
</br><br />
<br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). <h7>This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.</h7><br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its fuction <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis (less than 5% the cost of bisulfite sequencing).<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing data for samples marked *. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<br />
We varied induction conditions, expecting one might be more optimal than our initial inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase was still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7><br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. <br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:</h7><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.<br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay can detect site specific methylation in vivo.<br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/6/60/Zf-102813.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: A ZF-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 (N) control has no T7 polymerase and no possibility of leaky expression. The linearized control (L) is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
</br><i>Figure 1: The zinc finger-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. MaGellin reported off target activity, irrespective of zinc finger binding (Figure 1). <h7>MaGellin is sensitive to off target effects</h7> because there is no background CpG methylation in E.coli and the plasmid is short compared to a mammalian genome. We are interested in examining this initial finding further to determine its reproducibility and relevance to previously published zinc finger-methylases.<br />
</br><br />
<br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). <h7>This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.</h7><br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its fuction <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis (less than 5% the cost of bisulfite sequencing).<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing data for samples marked *. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<br />
We varied induction conditions, expecting one might be more optimal than our initial inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase was still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7><br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. <br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:</h7><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.<br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay can detect site specific methylation in vivo.<br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/6/60/Zf-102813.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: A ZF-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 (N) control has no T7 polymerase and no possibility of leaky expression. The linearized control (L) is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
</br><i>Figure 1: The zinc finger-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. MaGellin reported off target activity, irrespective of zinc finger binding (Figure 1). MaGellin is sensitive to off target effects because there is no background CpG methylation in E.coli and the plasmid is short compared to a mammalian genome. We are interested in examining this initial finding further to determine its reproducibility and relevance to previously published zinc finger-methylases.<br />
</br><br />
<br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). <h7>This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.</h7><br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its fuction <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis (less than 5% the cost of bisulfite sequencing).<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing data for samples marked *. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<br />
We varied induction conditions, expecting one might be more optimal than our initial inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase was still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7><br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. <br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:</h7><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.<br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). <h7>This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.</h7><br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7><br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of systems including two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:</h7><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.<br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). <h7>This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.</h7><br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<div id="text"><br />
<div class = "textwrap"><br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7><br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of systems including two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
</br></br><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h7>The process further validated our MaGellin assay:</h7><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.<br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.<br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<div id="text"><br />
<div class = "textwrap"><br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.<br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of systems including two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
The process further validated our MaGellin assay:<br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.<br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.<br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<div id="text"><br />
<div class = "textwrap"><br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.<br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of systems including two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<br />
<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h6>The process further validated our MaGellin assay: </h6><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.<br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.<br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<div id="text"><br />
<div class = "textwrap"><br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.<br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of systems including two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h5>The process further validated our MaGellin assay: </h5><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.<br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.<br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<div id="text"><br />
<div class = "textwrap"><br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.<br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of systems including two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h3>The process further validated our MaGellin assay: </h3><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.<br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.<br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<div id="text"><br />
<div class = "textwrap"><br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.<br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of systems including two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
<h2>The process further validated our MaGellin assay: </h2><br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.<br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.<br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<div id="text"><br />
<div class = "textwrap"><br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.<br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of systems including two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
The process further validated our MaGellin assay: <br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.<br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.<br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<div id="text"><br />
<div class = "textwrap"><br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.<br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of systems including two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
</br></br><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
The process further validated our MaGellin assay: <br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with bisulfite sequencing that the TALE exhibited targeted inhibition of the methylase. This has serious implications for the multitude of TALE-effector systems that have recently been developed: the TALE can inhibit the effector if the linker length and distance between the TALE binding site and target site are not optimized. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the enzymatic activity of our novel dCas9-M.SssI and are now characterizing it further.<br />
<br />
</br><br />
</br><br />
<h1>Zinc Finger-M.SssI Fusion</h1><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.<br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<div id="text"><br />
<div class = "textwrap"><br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.<br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h1>Summary</h1><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of systems including two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
</br></br><br />
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<b><center><h1><br />
Future Directions<br />
</b></center></h1><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br></br><br />
</br><br />
<h4><b>Advantages of a TALE-methylase</b></h4><br />
<p>Our new TALE fusion is much more modular and easy to customize than the old zinc finger (Table 1). TALE construction is not heavily patented like zinc-finger design, and TALE’s are considerably cheaper to produce (Sanjana 2012 and Zhang 2011). They have made the zinc-fingers nearly obsolete as a tool for genetic engineering, including the creation of transgenic animal models (Tesson 2011, Sander 2011, Huang 2011, and Zhang 2011). We expect our new fusion protein could be translated to enable the creation of differentially methylated animal models, which would be revolutionary for epigenetic disease research (Klose 2006). In effect, this could transform epigenetics from a largely observational discipline to one of active intervention and manipulation, similar to the transition from early classical genetics to genetic engineering and synthetic biology.<br />
</br><br />
<br />
<h4><b>CRISPR System</b></h4><p>We also fused a methyltransferase to a dCas9 DNA binding domain from the CRISPR-Cas system. We modified our MaGellin vector to include a cassette that expresses the necessary guiding sgRNA1. The sgRNA can easily be swapped out to quickly re-target the Cas system, much more quickly and with less synthesis than it would take to re-target the TALE. We are intrigued by the prospect of optimizing the Cas system, which is considered to possibly have many advantages over the TALE (Gaj 2013). It has already been fused to transcription activators that similarly target promoters, so the strategy is theoretically sound (Bikard 2013). Particularly, it’s ability to multiplex and target multiple sites for methylation would be very useful (Wang 2013). However, many recent papers have described problems with Cas specificity that seriously limit its usefulness, so we expect the excitement around Cas may dim as scientists begin a rigorous re-evaluation of the comparison between Cas and TALE systems. We propose MaGellin as a simple, fast, and inexpensive assay to aid the development of superior Cas systems, as they are optimized by site-directed mutagenesis, directed evolution, or other methods. As outlined above and demonstrated with our TALE, fusing a methylase to the Cas serves as a detectable marker for its DNA binding.</br><br />
<h4><b>Split-Reconstitution Site-Specific Methylases</b></h4><br />
</br>Most previously published zinc finger-methylase studies have used the same two components as our TALE, the DNA binding domain linked to the methylase. In the noiseless MaGellin system, we were able to detect significant off target methylation at long induction times and high IPTG concentrations. This suggests these fusions still methylate in a non-specific manner once the binding sites are saturated. This issue must be harder to detect in mammalian systems. We are proposing the best strategy would be to split the methylase into subunits that only dimerize and activate at the target site when co-localized by their linked TALEs. This has been effective for TALE nucleases.<br />
</br>We are going to try this strategy by using the other multiple cloning site on the MaGellin plasmid, which we have so far used to express an sgRNA for the dCas9-M.SssI.<br />
<br />
<h4><b>Translational Potential</b></h4><br />
<p>It is worth noting that the TALE-methylase can be delivered to mammalian cells by adeno-associated vectors (AAV). This viral vector is the best currently available in terms of safety and efficiency (Daya 2008). Different serotypes have different cell tropisms, which can provide efficient cell-type targeting when used in conjunction with a cell-specific promoter (Ellis 2013). As we consider different model organisms, we may also want to try different methyltransferases. Importantly, the TALE, at 2.5kb long, can easily be packaged with a methyltransferase in an AAV vector, whereas a Cas would not fit (Konermann 2013).<br />
<br />
<h4><b>Epigenome Engineering is a Reality Today</b></h4><br />
<p>We were not the only synthetic biologists developing epigenetic engineering tools this summer. In August, we were excited to see George Church’s lab at Harvard had fused TALEs to histone modifiers, enabling another distinct form of epigenetic engineering (Konermann 2013). They delivered these TALE fusions to mammalian cells with AAV, which gives us confidence our system can be translated into more complex organisms. With their targeted histone modification and our targeted DNA methylation, it appears the era of serious epigenetic engineering efforts is now upon us. <br />
<br />
<h4><b>Transcriptional Silencing in Bacteria with Methylation</b></h4><br />
<p>At the regional jamboree, we were asked how precisely we could use CpG methylation to silence transcription in E.coli, which have no such endogenous mechanism. In response, we have designed an experiment using MeCP2, a DNA binding protein that only binds when its recognition site is methylated. We hope to use this protein to hinder the polymerase. So far, we have cloned and purified the protein, including a variant which was truncated to remove the DNA binding domain, to serve as our negative control.<br />
<br />
<div align=left><br />
<h4><b>Alternative to <i>ChIP.</i> </b></h4><br />
<ol><li>MaGellin can also be used to screen the binding specificity of transcription factors (TF) and other DNA binding domains, with methylation serving as an effective reporter for targeted binding. </li><br />
<br />
<li>By fusing a TF to the methyltransferase, its binding with the plasmid becomes easily detected by the associated methylation</li><br />
<br />
<li>In this situation, one would vary the “target site” to see which DNA sequences have the strongest binding to the TF</li><br />
<br />
<li>This technique would be significantly faster, simpler, and cheaper than a ChIP-based method</li><br />
</ol><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
<br />
<br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for testing the activity and specificity of site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylase's tendency to also methylate off-target sequences; and were only measuring site-specific methylation.<br />
</br><br />
</br><br />
<h4><b>Measuring Methylation</b></h4> Two techniques have traditionally been employed to measure DNA methylation: <br />
</br><br />
</br><br />
<i>Restriction Based.</i> The first, called Combined Bisulfite Restriction Analysis (COBRA), involves chemically converting unmethylated cytosines into uracils (a process called bisulfite conversion), while leaving methylated cytosines intact. Performing PCR that amplifies the region of interest leaves methylated cytosines intact and converts unmethylated cytosines to thymines (Figure 1). Samples are then digested using an enzyme that will only cut the unconverted (originally methylated) cytosines. The enzyme can no longer recognize unmethylated sites, as they are “TG” instead of “CG”. Even with the help of advanced algorithms, designing primers for this process is not always feasible, and the process needs to be optimized each time a new site is to be analyzed. Furthermore, the workflow takes several days, is expensive, and is not high throughput enough to accommodate screening libraries of candidate DNA-binding-domain-methylase fusion proteins. It has recently fallen out of favor because it is difficult to interpret and does not consider all CpG sites, but only ones which fall within a restriction enzyme’s recognition sequence. (Xiong 1997 and Li 2002). Our methylation assay, MaGellin, is also restriction-based but is much simpler than COBRA because it does not require bisulfite conversion of the DNA. This eliminates most of the problems that made COBRA unwieldy.<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/f/fe/Cobra_workflow.svg/776px-Cobra_workflow.svg.png" alt="COBRA Methylation Assay" width="400" ><br />
</br><br />
<figure><img border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/7/7f/Cobra_quantification.svg/776px-Cobra_quantification.svg.png" width="400" ><br />
</figure><br />
<br />
<figcaption><i> Figure 1: COBRA. This restriction based assay detects methylation at CpG sites that fall within restriction enzyme recognition sequences.</i></figcaption></figure></div><br />
</br><br />
</br><br />
<br />
</br><br />
<i>Sequencing Based.</i> The second, and more commonly employed technique, is bisulfite sequencing, which employs the same bisulfite conversion step previously described, and is immediately followed by sequencing. Unmethylated cytosines are read as thymines and methylated cytosines are read as cytosines. Comparing converted and unconverted sequences reveals the methylation pattern with high resolution. Despite its advantages, this method is time consuming and can become very expensive as more and more constructs are screened for activity and specificity (Darst 2010). We determined to include standardized bisulfite sequencing primers on the MaGellin plasmid, so users would have the option of bisulfite sequencing after screening functional site-specific methylases with our quicker, less expensive, and intuitive digestion based assay.<br />
</br><br />
</br><br />
<center><h1>The MaGellin Methylation Assay</center><br />
</br><br />
</br><br />
<h4><b>Our Team’s Solution.</b></h4> In order to address the challenges associated with developing new site-specific methylase proteins, our team proposed several different strategies. First, we proposed a migration away from mammalian systems and into E. coli. E. coli does not have a native cytosine methylase, and therefore offers a noise-free environment for methylation studies. Any methylation of CpG sites in E Coli would be a product of a candidate engineered protein rather than the native organism. Second, we envisioned a modular one-plasmid system that can be employed for quickly and cheaply screening the activity and specificity of any DNA binding domain – methylase fusion protein. This plasmid-based methylation assay is called MaGellin.<br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/a/a5/Magellin_plasmid.png" height="400" alt="MaGellin"><figcaption><i>The MaGellin plasmid includes all the features needed to clone, express, and assay site-specific methylases.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Plasmid Features.</b> </h4>To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG methylase(M.SssI) with a generic linker sequence in the cloning site. Only a DNA binding domain needs to be cloned into the plasmid for a working fusion protein and assay, . This inherently standardizes MaGellin and minimizes the time a user of the assay needs to spend cloning.</li><br />
<li>Multiple cloning site downstream of T7 promoter for orthogonal expression of fusion protein in T7 Express competent E. coli.</li><br />
<li>Cloning site for a smaller DNA sequence, specific to the fusion protein being screened – named the “target site”, where the protein will bind. This can be the binding site for a Zinc Finger, TALE, CRISPR-Cas, or transcription factor.</li><br />
<li>AvaI restriction site 4 bases downstream of the target site – the AvaI restriction enzyme is blocked by methylated CpG sites, thus screening for site specific methylation becomes equivalent to screening for AvaI digestion</li><br />
<li>AvaI restriction site sufficiently further downstream of the target site – named the "off-target site". This site screens for non-specific DNA methylation as it is spatially removed from where the fusion protein binds to the plasmid.</li><br />
<li>XbaI site for linearization of the plasmid. Linearizing the plasmid simplifies analysis of the AvaI digestion by gel electrophoresis.</li><br />
<li>Validated bisulfite conversion primer binding sites, so users do not need to go through the time-consuming primer design process if they choose to complement MaGellin’s results with bisulfite sequencing for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>sgRNA cloning site for users who want to target a CRISPR-Cas binding domain. The sgRNA is constitutively expressed and can be swapped by restriction digest.</li><br />
<li>Kanamycin resistance as a selection marker.</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<h4><b>Noiseless Chassis.</b></h4> After cloning this plasmid, we faced the challenge of choosing the correct cell line for the assay. We chose to transform into T7 Express cells for several reasons:<br />
<ol><br />
<li>T7 RNA Polymerase in the lac operon allows us to turn on expression of fusion protein after induction with IPTG</li><br />
<li>In the T7 Express cell line, genes for several restriction enzymes known to target methylated DNA are knocked out (McrA-, McrBC-, EcoBr-m-, Mrr-). This ensures that our assay plasmid is not cleaved in vivo. Results are difficult, if not impossible, to interpret in the commonly used BL21 cell line.</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<h4><b>MaGellin Workflow.</b></h4> <br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<b>Assemble</b> the MaGellin backbone together with a DNA-binding protein and target sequence of your choosing.<br />
<li>Digest BBa_K1128001 (the MaGellin backbone) and BBa_K1128002 (the linker-M.ssI construct) with EcoRI and PstI.<br />
</li><br />
<li>Ligate K1128002 into the K1128001 backbone. </li><br />
<li>PCR amplify your DNA-binding protein of choice. In order to keep everything in frame, use the following 5’ extensions on the PCR primers:<ol><br />
<li> Forward: CAGGAGGAATTC[ATG] (add start codon only if not included in gene).</li><br />
<li> Reverse: CTCTAGAAGCGGC (make sure to remove the stop codon). </li> </ol><br />
<li> Use EcoRI and XbaI to ligate the DNA-binding protein into the MaGellin backbone, fusing it in frame to the linker-M.sssI construct.</li><br />
<li> Clone in your target sequence using BamHI and XhoI.</li><br />
<b>Methylate</b> the MaGellin plasmid <i>in vivo</i>.<ol><br />
<li>Transform the completed MaGellin plasmid into T7 Express. </li><br />
<li>Induce culture with 1 mM IPTG.</li><br />
<li>Incubate in a shaker at 37C for 5 hours.</li><br />
<li>Miniprep to isolate the plasmid.</li></ol><br />
<b>Digest</b> the methylated plasmid.<ol><br />
<li> Digest 600 ng of miniprep DNA in a 15 uL reaction with 10 U of both XbaI and AvaI.</li><br />
<li> Incubate reaction for 1 hour at 37C.</li></ol><br />
<b>Analyze</b> the data using the MaGellin Software Package.<br />
<li>Run the entire digestion reaction on a 1% agarose gel.</li><br />
<li>Take a photo of the gel.</li><br />
<li>Upload and analyze the gel photo using the MaGellin Software Package. </li><ol><br />
<li>Look for 3 distinct band patterns that correspond to specific and interpretable methylation outcomes.</li><ol><br />
<li>The presence of large one band corresponds to non-site-specific DNA methylation (AvaI was blocked at both the target and off target sites, and thus only XbaI cut the plasmid)</li><br />
<li>The presence of two bands corresponds to site-specific DNA methylation (AvaI was only blocked at the target site, thus AvaI cut in the off target site and XbaI cut the plasmid)</li><br />
<li>The presence of three bands corresponds to no DNA methylation – or an inactive fusion protein (AvaI was not blocked at either the target or off target sites and XbaI cut the plasmid) </li></ol><br />
</ol><br />
<br />
</br><br />
</br><br />
<br />
<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/a/ae/Workflow_Schematics.png" alt="Workflow" width="600" height="1000"><figcaption><i>Figure 2: The full workflow to use MaGellin, available from the BioBrick registry.</i></figcaption></figure></div><br />
</br><br />
</br><br />
<br />
<br />
<br />
<br />
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<center><object data="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf" type="application/x-shockwave-flash" width="600px" height="600px"><br />
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<param name="movie" value="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf"><br />
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<figcaption><i>Figure 3: Interactive animation of MaGellin workflow</i></figcaption><br />
</center><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
<br />
<br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for testing the activity and specificity of site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylase's tendency to also methylate off-target sequences; and were only measuring site-specific methylation.<br />
</br><br />
</br><br />
<h4><b>Measuring Methylation</b></h4> Two techniques have traditionally been employed to measure DNA methylation: <br />
</br><br />
</br><br />
<i>Restriction Based.</i> The first, called Combined Bisulfite Restriction Analysis (COBRA), involves chemically converting unmethylated cytosines into uracils (a process called bisulfite conversion), while leaving methylated cytosines intact. Performing PCR that amplifies the region of interest leaves methylated cytosines intact and converts unmethylated cytosines to thymines (Figure 1). Samples are then digested using an enzyme that will only cut the unconverted (originally methylated) cytosines. The enzyme can no longer recognize unmethylated sites, as they are “TG” instead of “CG”. Even with the help of advanced algorithms, designing primers for this process is not always feasible, and the process needs to be optimized each time a new site is to be analyzed. Furthermore, the workflow takes several days, is expensive, and is not high throughput enough to accommodate screening libraries of candidate DNA-binding-domain-methylase fusion proteins. It has recently fallen out of favor because it is difficult to interpret and does not consider all CpG sites, but only ones which fall within a restriction enzyme’s recognition sequence. (Xiong 1997 and Li 2002). Our methylation assay, MaGellin, is also restriction-based but is much simpler than COBRA because it does not require bisulfite conversion of the DNA. This eliminates most of the problems that made COBRA unwieldy.<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/f/fe/Cobra_workflow.svg/776px-Cobra_workflow.svg.png" alt="COBRA Methylation Assay" width="400" ><br />
</br><br />
<figure><img border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/7/7f/Cobra_quantification.svg/776px-Cobra_quantification.svg.png" width="400" ><br />
</figure><br />
<br />
<figcaption><i> Figure 1: COBRA. This restriction based assay detects methylation at CpG sites that fall within restriction enzyme recognition sequences.</i></figcaption></figure></div><br />
</br><br />
</br><br />
<br />
</br><br />
<i>Sequencing Based.</i> The second, and more commonly employed technique, is bisulfite sequencing, which employs the same bisulfite conversion step previously described, and is immediately followed by sequencing. Unmethylated cytosines are read as thymines and methylated cytosines are read as cytosines. Comparing converted and unconverted sequences reveals the methylation pattern with high resolution. Despite its advantages, this method is time consuming and can become very expensive as more and more constructs are screened for activity and specificity (Darst 2010). We determined to include standardized bisulfite sequencing primers on the MaGellin plasmid, so users would have the option of bisulfite sequencing after screening functional site-specific methylases with our quicker, less expensive, and intuitive digestion based assay.<br />
</br><br />
</br><br />
<center><h1>The MaGellin Methylation Assay</center><br />
</br><br />
</br><br />
<h4><b>Our Team’s Solution.</b></h4> In order to address the challenges associated with developing new site-specific methylase proteins, our team proposed several different strategies. First, we proposed a migration away from mammalian systems and into E. coli. E. coli does not have a native cytosine methylase, and therefore offers a noise-free environment for methylation studies. Any methylation of CpG sites in E Coli would be a product of a candidate engineered protein rather than the native organism. Second, we envisioned a modular one-plasmid system that can be employed for quickly and cheaply screening the activity and specificity of any DNA binding domain – methylase fusion protein. This plasmid-based methylation assay is called MaGellin.<br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/a/a5/Magellin_plasmid.png" height="400" alt="MaGellin"><figcaption><i>The MaGellin plasmid includes all the features needed to clone, express, and assay site-specific methylases.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Plasmid Features.</b> </h4>To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG methylase(M.SssI) with a generic linker sequence in the cloning site. Only a DNA binding domain needs to be cloned into the plasmid for a working fusion protein and assay, . This inherently standardizes MaGellin and minimizes the time a user of the assay needs to spend cloning.</li><br />
<li>Multiple cloning site downstream of T7 promoter for orthogonal expression of fusion protein in T7 Express competent E. coli.</li><br />
<li>Cloning site for a smaller DNA sequence, specific to the fusion protein being screened – named the “target site”, where the protein will bind. This can be the binding site for a Zinc Finger, TALE, CRISPR-Cas, or transcription factor.</li><br />
<li>AvaI restriction site 4 bases downstream of the target site – the AvaI restriction enzyme is blocked by methylated CpG sites, thus screening for site specific methylation becomes equivalent to screening for AvaI digestion</li><br />
<li>AvaI restriction site sufficiently further downstream of the target site – named the "off-target site". This site screens for non-specific DNA methylation as it is spatially removed from where the fusion protein binds to the plasmid.</li><br />
<li>XbaI site for linearization of the plasmid. Linearizing the plasmid simplifies analysis of the AvaI digestion by gel electrophoresis.</li><br />
<li>Validated bisulfite conversion primer binding sites, so users do not need to go through the time-consuming primer design process if they choose to complement MaGellin’s results with bisulfite sequencing for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>sgRNA cloning site for users who want to target a CRISPR-Cas binding domain. The sgRNA is constitutively expressed and can be swapped by restriction digest.</li><br />
<li>Kanamycin resistance as a selection marker.</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<h4><b>Noiseless Chassis.</b></h4> After cloning this plasmid, we faced the challenge of choosing the correct cell line for the assay. We chose to transform into T7 Express cells for several reasons:<br />
<ol><br />
<li>T7 RNA Polymerase in the lac operon allows us to turn on expression of fusion protein after induction with IPTG</li><br />
<li>In the T7 Express cell line, genes for several restriction enzymes known to target methylated DNA are knocked out (McrA-, McrBC-, EcoBr-m-, Mrr-). This ensures that our assay plasmid is not cleaved in vivo. Results are difficult, if not impossible, to interpret in the commonly used BL21 cell line.</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<h4><b>MaGellin Workflow.</b></h4> <br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<b>Assemble</b> the MaGellin backbone together with a DNA-binding protein and target sequence of your choosing.<br />
<li>Digest BBa_K1128001 (the MaGellin backbone) and BBa_K1128002 (the linker-M.ssI construct) with EcoRI and PstI.<br />
</li><br />
<li>Ligate K1128002 into the K1128001 backbone. </li><br />
<li>PCR amplify your DNA-binding protein of choice. In order to keep everything in frame, use the following 5’ extensions on the PCR primers:<ol><br />
<li> Forward: CAGGAGGAATTC[ATG] (add start codon only if not included in gene).</li><br />
<li> Reverse: CTCTAGAAGCGGC (make sure to remove the stop codon). </li> </ol><br />
<li> Use EcoRI and XbaI to ligate the DNA-binding protein into the MaGellin backbone, fusing it in frame to the linker-M.sssI construct.</li><br />
<li> Clone in your target sequence using BamHI and XhoI.</li><br />
<b>Methylate</b> the MaGellin plasmid <i>in vivo</i>.<ol><br />
<li>Transform the completed MaGellin plasmid into T7 Express. </li><br />
<li>Induce culture with 1 mM IPTG.</li><br />
<li>Incubate in a shaker at 37C for 5 hours.</li><br />
<li>Miniprep to isolate the plasmid.</li></ol><br />
<b>Digest</b> the methylated plasmid.<ol><br />
<li> Digest 600 ng of miniprep DNA in a 15 uL reaction with 10 U of both XbaI and AvaI.</li><br />
<li> Incubate reaction for 1 hour at 37C.</li></ol><br />
<b>Analyze</b> the data using the MaGellin Software Package.<br />
<li>Run the entire digestion reaction on a 1% agarose gel.</li><br />
<li>Take a photo of the gel.</li><br />
<li>Upload and analyze the gel photo using the MaGellin Software Package. </li><ol><br />
<li>Look for 3 distinct band patterns that correspond to specific and interpretable methylation outcomes.</li><ol><br />
<li>The presence of large one band corresponds to non-site-specific DNA methylation (AvaI was blocked at both the target and off target sites, and thus only XbaI cut the plasmid)</li><br />
<li>The presence of two bands corresponds to site-specific DNA methylation (AvaI was only blocked at the target site, thus AvaI cut in the off target site and XbaI cut the plasmid)</li><br />
<li>The presence of three bands corresponds to no DNA methylation – or an inactive fusion protein (AvaI was not blocked at either the target or off target sites and XbaI cut the plasmid) </li></ol><br />
</ol><br />
<br />
</br><br />
</br><br />
<br />
<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/a/ae/Workflow_Schematics.png" alt="Workflow" width="600" height="1000"><figcaption><i>Figure 2: The full workflow to use MaGellin, available from the BioBrick registry.</i></figcaption></figure></div><br />
</br><br />
</br><br />
<br />
<br />
<br />
<br />
<br />
<center><object data="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf" type="application/x-shockwave-flash" width="600px" height="600px"><br />
<br />
<param name="movie" value="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf"><br />
</object><br />
<figcaption><i>Figure 3: Interactive animation of MaGellin workflow</i></figcaption><br />
</center><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center><a href="https://2013.igem.org/Team:Penn/MaGellinToolbox">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/AssayValidation">Next&#8594;</a></center><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
<br />
<br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for testing the activity and specificity of site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylase's tendency to also methylate off-target sequences; and were only measuring site-specific methylation.<br />
</br><br />
</br><br />
<h4><b>Measuring Methylation</b></h4> Two techniques have traditionally been employed to measure DNA methylation: <br />
</br><br />
</br><br />
<i>Restriction Based.</i> The first, called Combined Bisulfite Restriction Analysis (COBRA), involves chemically converting unmethylated cytosines into uracils (a process called bisulfite conversion), while leaving methylated cytosines intact. Performing PCR that amplifies the region of interest leaves methylated cytosines intact and converts unmethylated cytosines to thymines (Figure 1). Samples are then digested using an enzyme that will only cut the unconverted (originally methylated) cytosines. The enzyme can no longer recognize unmethylated sites, as they are “TG” instead of “CG”. Even with the help of advanced algorithms, designing primers for this process is not always feasible, and the process needs to be optimized each time a new site is to be analyzed. Furthermore, the workflow takes several days, is expensive, and is not high throughput enough to accommodate screening libraries of candidate DNA-binding-domain-methylase fusion proteins. It has recently fallen out of favor because it is difficult to interpret and does not consider all CpG sites, but only ones which fall within a restriction enzyme’s recognition sequence. (Xiong 1997 and Li 2002). Our methylation assay, MaGellin, is also restriction-based but is much simpler than COBRA because it does not require bisulfite conversion of the DNA. This eliminates most of the problems that made COBRA unwieldy.<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/f/fe/Cobra_workflow.svg/776px-Cobra_workflow.svg.png" alt="COBRA Methylation Assay" width="400" ><br />
</br><br />
<figure><img border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/7/7f/Cobra_quantification.svg/776px-Cobra_quantification.svg.png" width="400" ><br />
</figure><br />
<br />
<figcaption><i> Figure 1: COBRA. This restriction based assay detects methylation at CpG sites that fall within restriction enzyme recognition sequences.</i></figcaption></figure></div><br />
</br><br />
</br><br />
<br />
</br><br />
<i>Sequencing Based.</i> The second, and more commonly employed technique, is bisulfite sequencing, which employs the same bisulfite conversion step previously described, and is immediately followed by sequencing. Unmethylated cytosines are read as thymines and methylated cytosines are read as cytosines. Comparing converted and unconverted sequences reveals the methylation pattern with high resolution. Despite its advantages, this method is time consuming and can become very expensive as more and more constructs are screened for activity and specificity (Darst 2010). We determined to include standardized bisulfite sequencing primers on the MaGellin plasmid, so users would have the option of bisulfite sequencing after screening functional site-specific methylases with our quicker, less expensive, and intuitive digestion based assay.<br />
</br><br />
</br><br />
<center><h1>The MaGellin Methylation Assay</center><br />
</br><br />
</br><br />
<h4><b>Our Team’s Solution.</b></h4> In order to address the challenges associated with developing new site-specific methylase proteins, our team proposed several different strategies. First, we proposed a migration away from mammalian systems and into E. coli. E. coli does not have a native cytosine methylase, and therefore offers a noise-free environment for methylation studies. Any methylation of CpG sites in E Coli would be a product of a candidate engineered protein rather than the native organism. Second, we envisioned a modular one-plasmid system that can be employed for quickly and cheaply screening the activity and specificity of any DNA binding domain – methylase fusion protein. This plasmid-based methylation assay is called MaGellin.<br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/a/a5/Magellin_plasmid.png" height="400" alt="MaGellin"><figcaption><i>The MaGellin plasmid includes all the features needed to clone, express, and assay site-specific methylases.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Plasmid Features.</b> </h4>To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG methylase(M.SssI) with a generic linker sequence in the cloning site. Only a DNA binding domain needs to be cloned into the plasmid for a working fusion protein and assay, . This inherently standardizes MaGellin and minimizes the time a user of the assay needs to spend cloning.</li><br />
<li>Multiple cloning site downstream of T7 promoter for orthogonal expression of fusion protein in T7 Express competent E. coli.</li><br />
<li>Cloning site for a smaller DNA sequence, specific to the fusion protein being screened – named the “target site”, where the protein will bind. This can be the binding site for a Zinc Finger, TALE, CRISPR-Cas, or transcription factor.</li><br />
<li>AvaI restriction site 4 bases downstream of the target site – the AvaI restriction enzyme is blocked by methylated CpG sites, thus screening for site specific methylation becomes equivalent to screening for AvaI digestion</li><br />
<li>AvaI restriction site sufficiently further downstream of the target site – named the "off-target site". This site screens for non-specific DNA methylation as it is spatially removed from where the fusion protein binds to the plasmid.</li><br />
<li>XbaI site for linearization of the plasmid. Linearizing the plasmid simplifies analysis of the AvaI digestion by gel electrophoresis.</li><br />
<li>Validated bisulfite conversion primer binding sites, so users do not need to go through the time-consuming primer design process if they choose to complement MaGellin’s results with bisulfite sequencing for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>sgRNA cloning site for users who want to target a CRISPR-Cas binding domain. The sgRNA is constitutively expressed and can be swapped by restriction digest.</li><br />
<li>Kanamycin resistance as a selection marker.</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<h4><b>Noiseless Chassis.</b></h4> After cloning this plasmid, we faced the challenge of choosing the correct cell line for the assay. We chose to transform into T7 Express cells for several reasons:<br />
<ol><br />
<li>T7 RNA Polymerase in the lac operon allows us to turn on expression of fusion protein after induction with IPTG</li><br />
<li>In the T7 Express cell line, genes for several restriction enzymes known to target methylated DNA are knocked out (McrA-, McrBC-, EcoBr-m-, Mrr-). This ensures that our assay plasmid is not cleaved in vivo. Results are difficult, if not impossible, to interpret in the commonly used BL21 cell line.</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<h4><b>MaGellin Workflow.</b></h4> <br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<b>Assemble</b> the MaGellin backbone together with a DNA-binding protein and target sequence of your choosing.<br />
<li>Digest BBa_K1128001 (the MaGellin backbone) and BBa_K1128002 (the linker-M.ssI construct) with EcoRI and PstI.<br />
</li><br />
<li>Ligate K1128002 into the K1128001 backbone. </li><br />
<li>PCR amplify your DNA-binding protein of choice. In order to keep everything in frame, use the following 5’ extensions on the PCR primers:<ol><br />
<li> Forward: CAGGAGGAATTC[ATG] (add start codon only if not included in gene).</li><br />
<li> Reverse: CTCTAGAAGCGGC (make sure to remove the stop codon). </li> </ol><br />
<li> Use EcoRI and XbaI to ligate the DNA-binding protein into the MaGellin backbone, fusing it in frame to the linker-M.sssI construct.</li><br />
<li> Clone in your target sequence using BamHI and XhoI.</li><br />
<b>Methylate</b> the MaGellin plasmid <i>in vivo</i>.<ol><br />
<li>Transform the completed MaGellin plasmid into T7 Express. </li><br />
<li>Induce culture with 1 mM IPTG.</li><br />
<li>Incubate in a shaker at 37C for 5 hours.</li><br />
<li>Miniprep to isolate the plasmid.</li></ol><br />
<b>Digest</b> the methylated plasmid.<ol><br />
<li> Digest 600 ng of miniprep DNA in a 15 uL reaction with 10 U of both XbaI and AvaI.</li><br />
<li> Incubate reaction for 1 hour at 37C.</li></ol><br />
<b>Analyze</b> the data using the MaGellin Software Package.<br />
<li>Run the entire digestion reaction on a 1% agarose gel.</li><br />
<li>Take a photo of the gel.</li><br />
<li>Upload and analyze the gel photo using the MaGellin Software Package. </li><ol><br />
<li>Look for 3 distinct band patterns that correspond to specific and interpretable methylation outcomes.</li><ol><br />
<li>The presence of large one band corresponds to non-site-specific DNA methylation (AvaI was blocked at both the target and off target sites, and thus only XbaI cut the plasmid)</li><br />
<li>The presence of two bands corresponds to site-specific DNA methylation (AvaI was only blocked at the target site, thus AvaI cut in the off target site and XbaI cut the plasmid)</li><br />
<li>The presence of three bands corresponds to no DNA methylation – or an inactive fusion protein (AvaI was not blocked at either the target or off target sites and XbaI cut the plasmid) </li></ol><br />
</ol><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
The process further validated our MaGellin assay: <br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with bisulfite sequencing that the TALE exhibited targeted inhibition of the methylase. This has serious implications for the multitude of TALE-effector systems that have recently been developed: the TALE can inhibit the effector if the linker length and distance between the TALE binding site and target site are not optimized. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the enzymatic activity of our novel dCas9-M.SssI and are now characterizing it further.<br />
<br />
</br><br />
</br><br />
<h4>Zinc Finger-M.SssI Fusion</h4><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.<br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
<div id="text"><br />
<div class = "textwrap"><br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.<br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h4>Summary</h4><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of systems including two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
The process further validated our MaGellin assay: <br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with bisulfite sequencing that the TALE exhibited targeted inhibition of the methylase. This has serious implications for the multitude of TALE-effector systems that have recently been developed: the TALE can inhibit the effector if the linker length and distance between the TALE binding site and target site are not optimized. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the enzymatic activity of our novel dCas9-M.SssI and are now characterizing it further.<br />
<br />
</br><br />
</br><br />
<h4>Zinc Finger-M.SssI Fusion</h4><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
<br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
<br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.<br />
</br><br />
</br><br />
<br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
</br><br />
<div id="text"><br />
<div class = "textwrap"><br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.<br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h4>Summary</h4><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of systems including two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
</br></br><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
The process further validated our MaGellin assay: <br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with bisulfite sequencing that the TALE exhibited targeted inhibition of the methylase. This has serious implications for the multitude of TALE-effector systems that have recently been developed: the TALE can inhibit the effector if the linker length and distance between the TALE binding site and target site are not optimized. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the enzymatic activity of our novel dCas9-M.SssI and are now characterizing it further.<br />
<br />
</br><br />
</br><br />
<h4>Zinc Finger-M.SssI Fusion</h4><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
</br><br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
</br><br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
</br><br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.<br />
</br><br />
</br><br />
</br><br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
</br><br />
<div id="text"><br />
<div class = "textwrap"><br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.<br />
</br><br />
</br><br />
<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.<br />
</br></br><br />
<h4>Summary</h4><br />
</br><br />
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.<br />
<br />
<p>We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of systems including two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
</br></br><br />
<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
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</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
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Early on, it became clear that there was no standardized assay for testing the activity and specificity of site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylase's tendency to also methylate off-target sequences; and were only measuring site-specific methylation.<br />
</br><br />
</br><br />
<h4><b>Measuring Methylation</b></h4> Two techniques have traditionally been employed to measure DNA methylation: <br />
</br><br />
</br><br />
<i>Restriction Based.</i> The first, called Combined Bisulfite Restriction Analysis (COBRA), involves chemically converting unmethylated cytosines into uracils (a process called bisulfite conversion), while leaving methylated cytosines intact. Performing PCR that amplifies the region of interest leaves methylated cytosines intact and converts unmethylated cytosines to thymines (Figure 1). Samples are then digested using an enzyme that will only cut the unconverted (originally methylated) cytosines. The enzyme can no longer recognize unmethylated sites, as they are “TG” instead of “CG”. Even with the help of advanced algorithms, designing primers for this process is not always feasible, and the process needs to be optimized each time a new site is to be analyzed. Furthermore, the workflow takes several days, is expensive, and is not high throughput enough to accommodate screening libraries of candidate DNA-binding-domain-methylase fusion proteins. It has recently fallen out of favor because it is difficult to interpret and does not consider all CpG sites, but only ones which fall within a restriction enzyme’s recognition sequence. (Xiong 1997 and Li 2002). Our methylation assay, MaGellin, is also restriction-based but is much simpler than COBRA because it does not require bisulfite conversion of the DNA. This eliminates most of the problems that made COBRA unwieldy.<br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/f/fe/Cobra_workflow.svg/776px-Cobra_workflow.svg.png" alt="COBRA Methylation Assay" width="400" ><br />
</br><br />
<figure><img border="0" src="http://upload.wikimedia.org/wikipedia/commons/thumb/7/7f/Cobra_quantification.svg/776px-Cobra_quantification.svg.png" width="400" ><br />
</figure><br />
<br />
<figcaption><i> Figure 1: COBRA. This restriction based assay detects methylation at CpG sites that fall within restriction enzyme recognition sequences.</i></figcaption></figure></div><br />
</br><br />
</br><br />
<br />
</br><br />
<i>Sequencing Based.</i> The second, and more commonly employed technique, is bisulfite sequencing, which employs the same bisulfite conversion step previously described, and is immediately followed by sequencing. Unmethylated cytosines are read as thymines and methylated cytosines are read as cytosines. Comparing converted and unconverted sequences reveals the methylation pattern with high resolution. Despite its advantages, this method is time consuming and can become very expensive as more and more constructs are screened for activity and specificity (Darst 2010). We determined to include standardized bisulfite sequencing primers on the MaGellin plasmid, so users would have the option of bisulfite sequencing after screening functional site-specific methylases with our quicker, less expensive, and intuitive digestion based assay.<br />
</br><br />
</br><br />
<center><h1>The MaGellin Methylation Assay</center><br />
</br><br />
</br><br />
<h4><b>Our Team’s Solution.</b></h4> In order to address the challenges associated with developing new site-specific methylase proteins, our team proposed several different strategies. First, we proposed a migration away from mammalian systems and into E. coli. E. coli does not have a native cytosine methylase, and therefore offers a noise-free environment for methylation studies. Any methylation of CpG sites in E Coli would be a product of a candidate engineered protein rather than the native organism. Second, we envisioned a modular one-plasmid system that can be employed for quickly and cheaply screening the activity and specificity of any DNA binding domain – methylase fusion protein. This plasmid-based methylation assay is called MaGellin.<br />
</br><br />
</br><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/a/a5/Magellin_plasmid.png" height="400" alt="MaGellin"><figcaption><i>The MaGellin plasmid includes all the features needed to clone, express, and assay site-specific methylases.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Plasmid Features.</b> </h4>To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG methylase(M.SssI) with a generic linker sequence in the cloning site. Only a DNA binding domain needs to be cloned into the plasmid for a working fusion protein and assay, . This inherently standardizes MaGellin and minimizes the time a user of the assay needs to spend cloning.</li><br />
<li>Multiple cloning site downstream of T7 promoter for orthogonal expression of fusion protein in T7 Express competent E. coli.</li><br />
<li>Cloning site for a smaller DNA sequence, specific to the fusion protein being screened – named the “target site”, where the protein will bind. This can be the binding site for a Zinc Finger, TALE, CRISPR-Cas, or transcription factor.</li><br />
<li>AvaI restriction site 4 bases downstream of the target site – the AvaI restriction enzyme is blocked by methylated CpG sites, thus screening for site specific methylation becomes equivalent to screening for AvaI digestion</li><br />
<li>AvaI restriction site sufficiently further downstream of the target site – named the "off-target site". This site screens for non-specific DNA methylation as it is spatially removed from where the fusion protein binds to the plasmid.</li><br />
<li>XbaI site for linearization of the plasmid. Linearizing the plasmid simplifies analysis of the AvaI digestion by gel electrophoresis.</li><br />
<li>Validated bisulfite conversion primer binding sites, so users do not need to go through the time-consuming primer design process if they choose to complement MaGellin’s results with bisulfite sequencing for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>sgRNA cloning site for users who want to target a CRISPR-Cas binding domain. The sgRNA is constitutively expressed and can be swapped by restriction digest.</li><br />
<li>Kanamycin resistance as a selection marker.</li><br />
</ol><br />
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</br><br />
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<h4><b>Noiseless Chassis.</b></h4> After cloning this plasmid, we faced the challenge of choosing the correct cell line for the assay. We chose to transform into T7 Express cells for several reasons:<br />
<ol><br />
<li>T7 RNA Polymerase in the lac operon allows us to turn on expression of fusion protein after induction with IPTG</li><br />
<li>In the T7 Express cell line, genes for several restriction enzymes known to target methylated DNA are knocked out (McrA-, McrBC-, EcoBr-m-, Mrr-). This ensures that our assay plasmid is not cleaved in vivo. Results are difficult, if not impossible, to interpret in the commonly used BL21 cell line.</li><br />
</ol><br />
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<h4><b>MaGellin Workflow.</b></h4> <br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<b>Assemble</b> the MaGellin backbone together with a DNA-binding protein and target sequence of your choosing.<br />
<li>Digest BBa_K1128001 (the MaGellin backbone) and BBa_K1128002 (the linker-M.ssI construct) with EcoRI and PstI.<br />
</li><br />
<li>Ligate K1128002 into the K1128001 backbone. </li><br />
<li>PCR amplify your DNA-binding protein of choice. In order to keep everything in frame, use the following 5’ extensions on the PCR primers:<ol><br />
<li> Forward: CAGGAGGAATTC[ATG] (add start codon only if not included in gene).</li><br />
<li> Reverse: CTCTAGAAGCGGC (make sure to remove the stop codon). </li> </ol><br />
<li> Use EcoRI and XbaI to ligate the DNA-binding protein into the MaGellin backbone, fusing it in frame to the linker-M.sssI construct.</li><br />
<li> Clone in your target sequence using BamHI and XhoI.</li><br />
<b>Methylate</b> the MaGellin plasmid <i>in vivo</i>.<ol><br />
<li>Transform the completed MaGellin plasmid into T7 Express. </li><br />
<li>Induce culture with 1 mM IPTG.</li><br />
<li>Incubate in a shaker at 37C for 5 hours.</li><br />
<li>Miniprep to isolate the plasmid.</li></ol><br />
<b>Digest</b> the methylated plasmid.<ol><br />
<li> Digest 600 ng of miniprep DNA in a 15 uL reaction with 10 U of both XbaI and AvaI.</li><br />
<li> Incubate reaction for 1 hour at 37C.</li></ol><br />
<b>Analyze</b> the data using the MaGellin Software Package.<br />
<li>Run the entire digestion reaction on a 1% agarose gel.</li><br />
<li>Take a photo of the gel.</li><br />
<li>Upload and analyze the gel photo using the MaGellin Software Package. </li><ol><br />
<li>Look for 3 distinct band patterns that correspond to specific and interpretable methylation outcomes.</li><ol><br />
<li>The presence of large one band corresponds to non-site-specific DNA methylation (AvaI was blocked at both the target and off target sites, and thus only XbaI cut the plasmid)</li><br />
<li>The presence of two bands corresponds to site-specific DNA methylation (AvaI was only blocked at the target site, thus AvaI cut in the off target site and XbaI cut the plasmid)</li><br />
<li>The presence of three bands corresponds to no DNA methylation – or an inactive fusion protein (AvaI was not blocked at either the target or off target sites and XbaI cut the plasmid) </li></ol><br />
</ol><br />
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/a/ae/Workflow_Schematics.png" alt="Workflow" width="600" height="1000"><figcaption><i>Figure 2: The full workflow to use MaGellin, available from the BioBrick registry.</i></figcaption></figure></div><br />
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<header><h1><b><center>Methylase Characterization</center></b></h1></header><br />
</br><br />
</br><br />
For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
</br><br />
</br><br />
We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br><br />
The process further validated our MaGellin assay: <br />
</br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.<br />
</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with bisulfite sequencing that the TALE exhibited targeted inhibition of the methylase. This has serious implications for the multitude of TALE-effector systems that have recently been developed: the TALE can inhibit the effector if the linker length and distance between the TALE binding site and target site are not optimized. The MaGellin workflow is well suited to solve this optimization problem.<br />
</br>3. We demonstrated the enzymatic activity of our novel dCas9-M.SssI and are now characterizing it further.<br />
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</br><br />
</br><br />
<h4>Zinc Finger-M.SssI Fusion</h4><br />
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.<br />
</br><br />
</br><br />
</br><br />
<font color="red">SHOW ZINC FINGER DATA</font><br />
</br><br />
</br><i>Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br><br />
</br><br />
</br><br />
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).<br />
</br><br />
</br><br />
</br><br />
<h1>TALE-M.SssI Fusion</h1><br />
</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.<br />
</br><br />
</br><br />
<h4><center>TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay</center></h4><br />
</br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.</i></figcaption></figure></div><br />
</br><br />
</br><br />
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.<br />
</br><br />
</br><br />
<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4><br />
</br><br />
Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result. </br><br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div><br />
</br><br />
</br><br />
We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.<br />
</br><br />
</br><br />
</br><br />
<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4><br />
</br><br />
Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.<br />
</br<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div><br />
</br><br />
<div id="text"><br />
<div class = "textwrap"><br />
We varied induction conditions, expecting one might be more optimal than our previous inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase is still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site. <br />
</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Those are our future directions, optimization of the system will be quick and easy with MaGellin. <b>We would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</b><br />
</br><br />
<br />
</br></br><br />
<center><b>Bisulfite Sequencing Confirms TALE Binding<br />
</center></b><br />
</br><br />
<br />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/23/TaleIndOn102513-1.png" alt="Workflow" width="500px"><figcaption><i>Figure 5: MaGellin standard bisulfite sequencing primers were used to bisulfite sequence 500 bp including the target site. Rows are individual clones, circles are CpG sites, and distances reflect their distance in bp. Filled in circles are methylated CpGs. </i></figcaption></figure></div><br />
</br></br><br />
The next step for characterizing functional constructs with MaGellin is to use the <a href="https://2013.igem.org/Team:Penn/AssayValidation">standard bisulfite sequencing primers </a> for a higher resolution look at individual CpG methylation. We bisulfite sequenced the target site of the induced TALE-M.SssI with the TALE binding site present 4 nucleotides upstream of the target AvaI cut site. The data further validated the enzymatic activity of our TALE-M.SssI in the region of the TALE binding site. It was also the first demonstration that the MaGellin assay and standard primers can easily be used for bisulfite sequencing with a TOPO cloning kit with ~7 day turnaround. Interestingly, no clones were methylated at the CpG site that is within the AvaI site itself (Figure 5). <br />
</br></br><br />
This data, in combination with the varied induction conditions and COBRA results, led us to hypothesize a new model for the TALE’s mechanism that successfully explains each result. Although 4 nucleotides between the zinc finger binding site and the target cut site was optimal for a published zinc finger fusion with a short linker. That distance is too short for use with a TALE fusion with our linker length. The TALE is at least three times the size of the zinc finger and so TALE binding occludes that CpG from interacting with the methylase.</br></br><br />
We have now shown that the novel TALE-M.SssI binds to its binding site strongly, as it almost fully protected that site from the methylase for 24 hours (Figure 4). We have shown that it exhibits methylating activity (Figure 2). Perhaps most interestingly, we have demonstrated that the TALE is large enough to physically occlude neighboring nucleotides from access to its linked effector, which has significant consequences for the recently published slew of TALE fusions – including the TALE-histone methylases, TALE-histone demethylases, and TALE-DNA demethylases for epigenetic engineering. We expect the same result will hold for Cas9-effector fusions, and are in the process of validating that hypothesis. We have already cloned the first dCas9-methylase fusion and are going to characterize its activity.<br />
</br></br><br />
<h4>Summary</h4><br />
</br><br />
MaGellin was developed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our workflow.<br />
<p>To gain our new insight into a fundamental shortcoming of recently developed genome engineering tools, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, bisulfite sequencing, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.<br />
<p>Moreover, we have demonstrated the importance of studying the distance between the binding site and the target site, and shown the ideal distance will be very different between different DNA binding domains. This boils down to an optimization problem between choosing binding sites and linker lengths; this is exactly the sort of problem that the MaGellin system is designed to solve in a fast and affordable manner.<br />
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