Team:Penn/MethylaseOverview

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<header><h1><b><center>Site Specific Methylase</center></b></h1></header>
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<header><h1><b><center>Site-Specific Methylases</center></b></h1></header>
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Now that the MaGellin assay was validated for non-specific methylases, we were ready to test site-specific methylases. These are fusion proteins, a DNA binding domain linked to a methylase by a serine glycine chain. We used the prokaryotic methylase M.SssI for all of our studies (BBa_K1128000).  
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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.
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We wanted to first recapitulate published results with a zinc finger binding domain, and then characterize our two novel site-specific methylases: using the TALE and CRISPR/Cas binding domains.
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INSERT TABLE COMPARISON HERE
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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).  
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<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.
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<h1>Zinc Finger-M.SssI Fusion. </h>
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</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.
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SHOW ZINC FINGER DATA
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<center><IMG SRC="http://farm6.staticflickr.com/5478/10529350195_2c54c496e5.jpg" width="240" height="auto"><center></br></br>
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</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>
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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).
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<h1>TALE-M.SssI Fusion. </h>
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</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 quickly replacing zinc fingers. We performed a similar experiment with our TALE-M.SssI fusion, with and without the binding site present at the target cut site. We ran the gel and saw a significant effect on the methylation pattern. At first, the activity seemed similar to the zinc finger but it was not in agreement with our software’s predicted experimental outcome.
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<b><center>TALE-M.SssI actively methylates DNA</center></b>
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Latest revision as of 03:27, 29 October 2013

Penn iGEM

Site-Specific Methylases



For a detailed, graphical explanation of the MaGellin work flow, please download the MaGellin Workflow Specifications Sheet, which includes all of the steps in the MaGellin workflow.

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).

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.



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