Team:Penn/Project

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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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            <b><center><h1>
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Project Description
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The code of life is more than a sequence of A’s, C’s, T’s and G’s; epigenetic modifications, such as DNA methylation, are powerful and heritable regulators of gene expression. Targeted methyltransferases are enzymes that catalyze sequence-specific methylation – the most useful tool for engineering the epigenome. With a synthetic biology approach, we developed an assay to test targeted methyltransferases without expensive, time-consuming traditional methods. Our modular single-plasmid system allows methyltransferases to be easily cloned and tested via inexpensive digestion assays, quickly measuring the existence and extent of targeted methylation. Additionally, our plasmid contains standardized primer-binding sites for methylation-sensitive sequencing, and our E. coli chassis effectively eliminated noise associated with methylation studies. We are using this assay to characterize our novel targeted methyltransferases, which could be used to study epigenetic modifications. In the future, synthetic biologists could embrace these tools to explore the next frontier in engineering biological systems: the epigenome.</center>
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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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<p>For example, this is CopyCat: <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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  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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  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 />
 
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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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  <strong>Targeted Methylation:  the Possibilities</strong><br />
 
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  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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<p>MaGellin has several key features: </p>
 
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  <li>DNA  binding domain/methyltransferase can be swapped out</li>
 
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  <li>Easy  digest assays both components: methylation and targeting</li>
 
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  <li>Inducible  system for controlled expression of protein</li>
 
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  <li>Verified  working bisulfite sequencing primers</li>
 
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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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<p>The following table compares bisulfite sequencing (the  currently used standard for measuring DNA methylation) to Magellin.</p>
 
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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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    <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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    <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="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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    <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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    <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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    <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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<p><strong>Our team used  MaGellin to Characterize Existing and Novel Targeted Methyltransferases:</strong></p>
 
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Latest revision as of 03:50, 28 September 2013

modeling

Project Description


The code of life is more than a sequence of A’s, C’s, T’s and G’s; epigenetic modifications, such as DNA methylation, are powerful and heritable regulators of gene expression. Targeted methyltransferases are enzymes that catalyze sequence-specific methylation – the most useful tool for engineering the epigenome. With a synthetic biology approach, we developed an assay to test targeted methyltransferases without expensive, time-consuming traditional methods. Our modular single-plasmid system allows methyltransferases to be easily cloned and tested via inexpensive digestion assays, quickly measuring the existence and extent of targeted methylation. Additionally, our plasmid contains standardized primer-binding sites for methylation-sensitive sequencing, and our E. coli chassis effectively eliminated noise associated with methylation studies. We are using this assay to characterize our novel targeted methyltransferases, which could be used to study epigenetic modifications. In the future, synthetic biologists could embrace these tools to explore the next frontier in engineering biological systems: the epigenome.

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