Team:Penn/Project

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    <title>Project</title>
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<img src="http://upload.wikimedia.org/wikipedia/en/d/d6/IGEM_official_logo.png" id="igem"/><!--igem logo-->
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<img src="http://collegediabetesnetwork.org/wp-content/uploads/2012/07/UPenn_logo1.png" id="penn"/> <!--penn logo-->
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    <div class="section-title">team</div>
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              <p><strong>Background</strong><br />
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  DNA methylation impacts many cellular processes including cell  differentiation, genomic imprinting, DNA replication, X chromosome  inactivation, and suppression of unneeded transcription from oncogenes. <br />
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  <img src="http://s22.postimg.org/4td20mp1t/first.png" alt="" width="181" height="88" hspace="12" align="left" /><br />
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  <img src="http://s23.postimg.org/lqyr0fo17/second.png" alt="" width="174" height="48" hspace="12" align="left" /></p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<br>
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<p>For example, this is CopyCat: <br />
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<br>
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<img src="http://s16.postimg.org/ycopzivdx/third.png" alt="" width="169" height="255" hspace="12" align="left" /></p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<p>                         <br />
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  </p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<br>
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<br>
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<br>
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<br>
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<p>CopyCat is the first cloned pet,  born in December 2001 (Shin, Taeyoung, et al. (2002)), and this is Rainbow:<br />
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  <img src="http://s22.postimg.org/lvoq52edt/fourth.png" alt="" width="173" height="156" /><br />
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  Rainbow is CopyCat&rsquo;s genetic  donor; note that they have totally different fur patterns – this is due to  their epigenetic differences, particularly a phenomenon called X-Chromosome  inactivation. This is a dosage compensation mechanism for females wherein one X  chromosome in each cell is randomly selected for inactivation by DNA  methylation and other modifications.</p>
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<p><br />
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  DNA methylation also has another important effect in  mammalian cells; methylation of CpG sites can function to repress gene  expression, as shown where methylation prevents binding of DNA polymerase  upstream of a promoter, which is then silenced. <br />
 +
  <img width="369" height="237" src="http://s21.postimg.org/dyqgrnw7r/fifth.png" align="left" hspace="12" alt="Description: Screen Shot 2013-08-26 at 2.20.24 PM.png" /></p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<br>
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<br>
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<br>
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<br>
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<br>
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<p>Clearly, DNA methylation is one of nature&rsquo;s most powerful  mechanisms for transcriptional regulation, yet synthetic biologists don&rsquo;t talk  about it. Penn iGEM is adding synthetic biology as a tool for epigenetics  researchers; we are providing researchers with the tools they need to safely  and precisely engineer the epigenome. </p>
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<p><br />
 +
  <strong>Targeted Methylation:  the Possibilities</strong><br />
 +
  If the tools existed to allow researchers to target specific  DNA sequences for selective methylation, they could be used to inactivate  strong promoters (permanent repression without gene knockout), repress  overactive oncogenes, inactivate excess chromosomes, correct hypomethylation in  imprinting disorders and even control differentiation in stem cells. <br />
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  Our team aimed to address this need by creating a target  methylation toolkit which includes a modular plasmid that enables fast, cheap,  and simple screening of targeted methyltransferases. </p>
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<p><strong>MaGellin: A one plasmid system for screening targeted  methyltransferases</strong><br />
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  <img src="http://s21.postimg.org/vtvwmdh3r/sixth.png" alt="" width="290" height="290" hspace="12" align="left" /></p>
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<p>&nbsp;</p>
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<br clear="all" />
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<p>&nbsp;</p>
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<p>MaGellin has several key features: </p>
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<ol>
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  <li>DNA  binding domain/methyltransferase can be swapped out</li>
 +
  <li>Easy  digest assays both components: methylation and targeting</li>
 +
  <li>Inducible  system for controlled expression of protein</li>
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  <li>Verified  working bisulfite sequencing primers</li>
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</ol>
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<p>Restriction digest of miniprep screens targeted  methyltransferase efficacy<br />
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  <img width="139" height="446" src="http://s12.postimg.org/fqgv07s25/seventh.png" alt="Description: One Plasmid System.jpg" /></p>
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<p>This assay gives Results 3 hours after miniprep. Bisulfite  sequencing would take 5 days and much more money.</p>
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<br clear="all" />
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<p>The following table compares bisulfite sequencing (the  currently used standard for measuring DNA methylation) to Magellin.</p>
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<table border="1" cellspacing="0" cellpadding="0" width="602">
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  <tr>
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    <td width="200" valign="top"></td>
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    <td width="201" valign="top"><p><strong>Bisulfite Sequencing</strong></p></td>
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    <td width="201" valign="top"><p><strong>Modular Plasmid</strong></p></td>
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  </tr>
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  <tr>
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    <td width="200" valign="top"><p>Time after Sample Collection</p></td>
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    <td width="201" valign="top" bgcolor="#FF0000"><p>5 days</p></td>
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    <td width="201" valign="top" bgcolor="#00FF00"><p>2 hours</p></td>
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  </tr>
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  <tr>
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    <td width="200" valign="top"><p>Cost</p></td>
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    <td width="201" valign="top" bgcolor="#FF0000"><p>$$$</p></td>
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    <td width="201" valign="top" bgcolor="#00FF00"><p>$</p></td>
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  </tr>
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  <tr>
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    <td width="200" valign="top"><p>Reports On-Target and Off-Target Effects</p></td>
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    <td width="201" valign="top" bgcolor="#00FF00"><p>Requires separate assays</p></td>
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    <td width="201" valign="top" bgcolor="#00FF00"><p>Can be assayed simultaneously</p></td>
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  </tr>
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  <tr>
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    <td width="200" valign="top"><p>Quantitative Measurement of Methylation</p></td>
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    <td width="201" valign="top" bgcolor="#00FF00"><p>Yes</p></td>
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    <td width="201" valign="top" bgcolor="#00FF00"><p>No</p></td>
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  </tr>
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  <tr>
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    <td width="200" valign="top"><p>Ease of Designing Sequencing Primers</p></td>
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    <td width="201" valign="top" bgcolor="#FF0000"><p>Difficult</p></td>
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    <td width="201" valign="top" bgcolor="#00FF00"><p>Primers already designed</p></td>
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  </tr>
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  <tr>
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    <td width="200" valign="top"><p>Ease of Cloning</p></td>
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    <td width="201" valign="top" bgcolor="#FF0000"><p>Must either co-transform or design cloning plan for    single-plasmid system</p></td>
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    <td width="201" valign="top" bgcolor="#00FF00"><p>Backbone with target is ready for cloning</p></td>
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  </tr>
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</table>
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<p><strong>Our team used  MaGellin to Characterize Existing and Novel Targeted Methyltransferases:</strong></p>
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Revision as of 21:06, 25 September 2013

Project

team

Background
DNA methylation impacts many cellular processes including cell differentiation, genomic imprinting, DNA replication, X chromosome inactivation, and suppression of unneeded transcription from oncogenes.

 

 


For example, this is CopyCat:

 

 

 

                        

 

 

 





CopyCat is the first cloned pet, born in December 2001 (Shin, Taeyoung, et al. (2002)), and this is Rainbow:

Rainbow is CopyCat’s genetic donor; note that they have totally different fur patterns – this is due to their epigenetic differences, particularly a phenomenon called X-Chromosome inactivation. This is a dosage compensation mechanism for females wherein one X chromosome in each cell is randomly selected for inactivation by DNA methylation and other modifications.


DNA methylation also has another important effect in mammalian cells; methylation of CpG sites can function to repress gene expression, as shown where methylation prevents binding of DNA polymerase upstream of a promoter, which is then silenced.
Description: Screen Shot 2013-08-26 at 2.20.24 PM.png

 

 

 

 

 

 






Clearly, DNA methylation is one of nature’s most powerful mechanisms for transcriptional regulation, yet synthetic biologists don’t talk about it. Penn iGEM is adding synthetic biology as a tool for epigenetics researchers; we are providing researchers with the tools they need to safely and precisely engineer the epigenome.


Targeted Methylation: the Possibilities
If the tools existed to allow researchers to target specific DNA sequences for selective methylation, they could be used to inactivate strong promoters (permanent repression without gene knockout), repress overactive oncogenes, inactivate excess chromosomes, correct hypomethylation in imprinting disorders and even control differentiation in stem cells.
Our team aimed to address this need by creating a target methylation toolkit which includes a modular plasmid that enables fast, cheap, and simple screening of targeted methyltransferases.

MaGellin: A one plasmid system for screening targeted methyltransferases

 


 

MaGellin has several key features:

  1. DNA binding domain/methyltransferase can be swapped out
  2. Easy digest assays both components: methylation and targeting
  3. Inducible system for controlled expression of protein
  4. Verified working bisulfite sequencing primers

Restriction digest of miniprep screens targeted methyltransferase efficacy
Description: One Plasmid System.jpg

This assay gives Results 3 hours after miniprep. Bisulfite sequencing would take 5 days and much more money.


The following table compares bisulfite sequencing (the currently used standard for measuring DNA methylation) to Magellin.

Bisulfite Sequencing

Modular Plasmid

Time after Sample Collection

5 days

2 hours

Cost

$$$

$

Reports On-Target and Off-Target Effects

Requires separate assays

Can be assayed simultaneously

Quantitative Measurement of Methylation

Yes

No

Ease of Designing Sequencing Primers

Difficult

Primers already designed

Ease of Cloning

Must either co-transform or design cloning plan for single-plasmid system

Backbone with target is ready for cloning

Our team used MaGellin to Characterize Existing and Novel Targeted Methyltransferases:

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