Team:Penn/FusionResults

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             <b><center><h1>
             <b><center><h1>
Results
Results
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<b>Creating a Novel Targeted Methylation Fusion Protein</b>
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<center><b>Creating a Novel Targeted Methylation Fusion Protein</b><br></center>
<i>Novel Enzyme. </i>Now that our assay was validated, we decided to create a new DNA binding domain – methyltransferase fusion protein that we thought would be more specific than the 30-year-old zinc finger. We created a novel TALE – M.SssI fusion protein and assayed its activity and specificity. <br><br>
<i>Novel Enzyme. </i>Now that our assay was validated, we decided to create a new DNA binding domain – methyltransferase fusion protein that we thought would be more specific than the 30-year-old zinc finger. We created a novel TALE – M.SssI fusion protein and assayed its activity and specificity. <br><br>
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SAME AS ABOVE FOR ZINC FINGER ASSAY<br><br>
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<img src="https://static.igem.org/mediawiki/2013/a/a6/TALE_Representative_Bands.jpg" width=600><br><br>
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<i>We ran our assay with the TALE Fusion, with or without the target site on the plasmid. We saw significantly different band patterns. The fusion did not cause off-target methylation when there was a target site included.</i><br><br>
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<img src="https://static.igem.org/mediawiki/2013/6/6f/TALE_Targeting_Graph_AV.png" width=600><br><br>
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<i>We quantified the gel with our MaGellin software</i><br><br>
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<i>Demonstrated Efficacy and Specificity.</i> We found that expression of the fusion TALE-Methyltransferase protein led to significant site-specific DNA methylation. To further convince ourselves of the positive result, we cut the TALE binding “target” site out of the MaGellin plasmid and found this effectively diminished methylation at that site. Off target methylation was minimal, as opposed to what we saw with the zinc-finger-methyltransferase.<br><br>
<i>Demonstrated Efficacy and Specificity.</i> We found that expression of the fusion TALE-Methyltransferase protein led to significant site-specific DNA methylation. To further convince ourselves of the positive result, we cut the TALE binding “target” site out of the MaGellin plasmid and found this effectively diminished methylation at that site. Off target methylation was minimal, as opposed to what we saw with the zinc-finger-methyltransferase.<br><br>
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<i>Advantages. </i>Our new TALE fusion is also much more modular and easy to customize than the old zinc finger. 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>
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<i>Advantages. </i>Our new TALE fusion is also much more modular and easy to customize than the old zinc finger. 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><center>
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<img src="https://static.igem.org/mediawiki/2013/f/f1/Zf_methyl_tale_thing.png" width=600><br>
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<i>Our TALE fusion has numerous advantages over the existing Zinc Finger fusions.</i><br><br>
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</center>
<i>Achievement. </i>This is the first easily customizable protein that targets CpG methylation to specific sequences; additionally, since CpG methylation is not native to E. coli, we have created a tool that modifies epigenetics in E. coli. Synthetic biologists could use such a tool to assess the effect of CpG methylation on bacterial promoters and maybe even discover an orthogonal method to silence constitutive promoters. We expect synthetic biologists will soon take it as fact that there are more than 4 nucleotides in DNA. Methylated cytosines have starkly different properties from unmethylated cytosines, and other bases can be methylated, too. We have heard that synthetic biologists are poets who write in DNA; we are proud to have expanded their alphabet.
<i>Achievement. </i>This is the first easily customizable protein that targets CpG methylation to specific sequences; additionally, since CpG methylation is not native to E. coli, we have created a tool that modifies epigenetics in E. coli. Synthetic biologists could use such a tool to assess the effect of CpG methylation on bacterial promoters and maybe even discover an orthogonal method to silence constitutive promoters. We expect synthetic biologists will soon take it as fact that there are more than 4 nucleotides in DNA. Methylated cytosines have starkly different properties from unmethylated cytosines, and other bases can be methylated, too. We have heard that synthetic biologists are poets who write in DNA; we are proud to have expanded their alphabet.
 +
<ol><br>
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<li>We have developed a novel TALE-M.SssI fusion protein that works better than existing equivalents</li>
 +
 +
<li>Our protein is more specific</li>
 +
 +
<li>TALE binding domains are easy to customize and tailor to target specific sequences at lower costs</li>
 +
<li>Synthetic biologists could use our protein to begin experimenting with an orthogonal level of control in E. coli – they can begin to engineer the epigenome
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</li>
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</ol>
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<center><a href="https://2013.igem.org/Team:Penn/FusionMotivation">&#8592;Previous  </a>
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<a href="https://2013.igem.org/Team:Penn/FusionFutureDirections"> Next&#8594;</a></center>
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<a href="https://2013.igem.org/Team:Penn/FusionFutureDirections">Next&#8594;</a></center>
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Latest revision as of 04:27, 22 October 2013

Penn iGEM

Results


Creating a Novel Targeted Methylation Fusion Protein
Novel Enzyme. Now that our assay was validated, we decided to create a new DNA binding domain – methyltransferase fusion protein that we thought would be more specific than the 30-year-old zinc finger. We created a novel TALE – M.SssI fusion protein and assayed its activity and specificity.



We ran our assay with the TALE Fusion, with or without the target site on the plasmid. We saw significantly different band patterns. The fusion did not cause off-target methylation when there was a target site included.



We quantified the gel with our MaGellin software

Demonstrated Efficacy and Specificity. We found that expression of the fusion TALE-Methyltransferase protein led to significant site-specific DNA methylation. To further convince ourselves of the positive result, we cut the TALE binding “target” site out of the MaGellin plasmid and found this effectively diminished methylation at that site. Off target methylation was minimal, as opposed to what we saw with the zinc-finger-methyltransferase.

Advantages. Our new TALE fusion is also much more modular and easy to customize than the old zinc finger. 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.


Our TALE fusion has numerous advantages over the existing Zinc Finger fusions.

Achievement. This is the first easily customizable protein that targets CpG methylation to specific sequences; additionally, since CpG methylation is not native to E. coli, we have created a tool that modifies epigenetics in E. coli. Synthetic biologists could use such a tool to assess the effect of CpG methylation on bacterial promoters and maybe even discover an orthogonal method to silence constitutive promoters. We expect synthetic biologists will soon take it as fact that there are more than 4 nucleotides in DNA. Methylated cytosines have starkly different properties from unmethylated cytosines, and other bases can be methylated, too. We have heard that synthetic biologists are poets who write in DNA; we are proud to have expanded their alphabet.

  1. We have developed a novel TALE-M.SssI fusion protein that works better than existing equivalents
  2. Our protein is more specific
  3. TALE binding domains are easy to customize and tailor to target specific sequences at lower costs
  4. Synthetic biologists could use our protein to begin experimenting with an orthogonal level of control in E. coli – they can begin to engineer the epigenome


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