http://2013.igem.org/wiki/index.php?title=Special:Contributions/Magaraci&feed=atom&limit=50&target=Magaraci&year=&month=2013.igem.org - User contributions [en]2024-03-29T15:39:15ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:Penn/AbstractTeam:Penn/Abstract2013-10-29T03:26:10Z<p>Magaraci: </p>
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<b><center><h1><br />
Project Overview<br />
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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 />
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</html></div>Magaracihttp://2013.igem.org/Team:Penn/AssayOverviewTeam:Penn/AssayOverview2013-10-29T02:49:09Z<p>Magaraci: </p>
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
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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.<br />
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<i>Click on the image below to play a step-by-step animation that details the process.</i><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 />
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<h4><b>Measuring Methylation</b></h4> Two techniques have traditionally been employed to measure DNA methylation: <br />
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<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 />
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<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 />
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<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 />
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<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 />
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<center><h1>The MaGellin Methylation Assay</center><br />
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<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 />
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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/a5/Magellin_plasmid.png" height="400" alt="MaGellin"><figcaption><i>Figure 2: The MaGellin plasmid includes all the features needed to clone, express, and assay site-specific methylases.</i></figcaption></figure></div><br />
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<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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<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 />
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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 />
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The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
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<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 />
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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 3: The full workflow to use MaGellin, available from the BioBrick registry.</i></figcaption></figure></div><br />
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<b><center><h1>Background Information</h1></center></b><br />
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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.<br />
</br><br />
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<h4><b>Epigenetics.</b></h4> 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 />
</br><br />
<h4><b>DNA Methylation.</b></h4> DNA methylation is one of the most prominent and powerful mechanisms of epigenetic control. In mammals, enzymes called methyltransferases catalyze the addition of methyl groups to cytosines in CpG sites (a cytosine next to a guanine); these sites are abundant throughout the human genome (Venter 2001). Methylated cytosines in CpG sites can block transcription factors from binding to DNA and repress gene expression (Jones 1998 and Nan 1998). Although epigenetic factors do not affect genotype, they do affect phenotype. Specific methylation patterns are necessary for healthy development and disruption of methylation patterns has been shown to lead to many diseases (Cooper 1988, Rideout III 1990, Baylin 1998, Jones 1999, and Amir 1999).<br />
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<b><center><h1>An Unmet Need</h1></center></b><br />
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<h4><b>Epigenetic Engineering.</b></h4> Synthetic biology largely involved engineering genetic networks in bacterial chasses at its inception, but there have been increasing efforts to engineer more complex mammalian systems. Yet, despite the dramatic effects that subtle epigenetic modifications can have on phenotype, there are no robust and well-characterized tools for engineering the epigenome. If synthetic biologists intend to successfully transition from bacterial to mammalian chasses, they must appreciate the epigenetic control of gene expression. An epigenetic toolbox for synthetic biology would enable the creation of engineered organisms that more closely rival their natural counterparts with regard to the subtlety and robustness of cellular control. We decided to make one of these tools, an enzyme that can direct DNA methylation to specific sequences.<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/b/b2/Transcriptional-Silencing.png" alt="In Vivo Methylation" width="600"><figcaption><i>Methylation-mediated transcriptional silencing could grant synthetic biologists access to an additional layer of genetic control.</i></figcaption></figure></div><br />
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<h4><b>Epigenetic Disease.</b></h4> Methylation abnormalities are linked to a wide range of diseases. Many types of cancer can be characterized by their DNA methylation profiles. In fact, DNA methylation has been called “the hallmark of cancer” (Syzf, 2004). Specifically, hypomethylation of oncogenes has been linked to tumorigenesis and loss of CpG methylation at specific sites has been implicated as a main cause of cancer. No drugs, approved or in testing, can restore methylation. We determined to engineer enzymes that are up to the task.<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/2013/7/78/Healthy-to-Cancer.png" alt="Hypomethylation" width="600"><figcaption><i>Site-specific methylation could potentially restore normal methylation levels to hypomethylated cells.</i></figcaption></figure></div><br />
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<b><center><h1>Existing Technologies</h1></center></b><br />
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<h4><b>Zinc-Finger Systems.</b> </h4>Some progress has been made towards developing a tool that can make methylate DNA in a controlled manner (Xu 1997, Carvin 2003, and van Steensel 2000). Methylases are the enzymes which catalyze DNA methylation, but they are not inherently targeted to any specific DNA sequence. Since the 1990s, zinc finger proteins that bind a given DNA target sequence have been fused to methylases in an attempt to create an enzyme capable of methylating predetermined DNA sequences (Xu 1997). Although these fusion proteins have been somewhat successful in directing and controlling DNA methylation, they are known to methylate “off-target” DNA sequences distinct from the region intended to be methylated, it is difficult to modify the zinc finger domain to target unique DNA sequences, and the protein engineering process is expensive (Li 2006, Papwort 2005, and Desjarlais 1992). For these reasons, zinc finger methyltransferase fusion proteins have not gained wide spread use in epigenetic studies, and have not been considered for therapeutic purposes. We recapitulated the results with published zinc finger fusions, but were eager to improve on these existing technologies.<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/2013/2/2d/Msssi.gif" alt="M.SssI" width="600" height="500"><figcaption><i>Molecular model of M.SssI (Generated using Phyre2)</i></figcaption></figure></div><br />
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<h4><b>TAL Effector Systems.</b></h4> The TALE is a more site specific and easily customizable DNA-binding domain than the zinc finger, which has increasingly been used in synthetic biology DNA targeting experiments. As we worked on our project this summer and fall, we were thrilled to see other labs thinking about epigenetic engineering. In the past few months, there has been published work on TALEs fused with histone methylases, histone demethylases, and DNA demethylases (Konermann 2013, Mendenhall 2013, Maedner 2013). The contribution of our TALE DNA methylase completes this initial suite of tools for epigenetic engineering.<br />
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<h4><b>Slowed Progress.</b></h4>DNA binding proteins that are more modular and specific to their target sequence than zinc-fingers, such as the TALE and CRISPR-Cas systems, have gained widespread attention for their utility in genetic studies, but they have not been leveraged for improved targeted methylation. Screening new methyltransferase fusions for activity and specificity is difficult and expensive, which could hamper protein-engineering efforts (Gaj 2013). So, we developed a new methylation assay, MaGellin, to accelerate development and cut costs. Then, we used MaGellin to prove the efficacy of the first TALE-methylase fusion.<br />
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<h4><b>Noise Problem.</b></h4>An additional challenge that makes the design and characterization of proteins which methylate specific DNA sequences is the signal:noise ratio in mammalian cells. With a large amount of CpG sites naturally methylated in mammalian cells, it is difficult to differentiate protein activity from background noise. However, CpG methylation is completely orthogonal to E.coli so we developed our methylation assay, MaGellin, in this bacterial chassis.<br />
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<h4><b>Lack of a Standarized Assay.</b></h4>The most pressing challenge, however, was the lack of a standardized assay for the activity of site-specific methylases that was inexpensive, fast, robust, and easy to use. So, the first element of our project and our most important contribution to the community was the development of the MaGellin assay.<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 />
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<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 />
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</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 />
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</br><br />
</br><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/2013/0/00/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 />
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</br><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/2013/5/58/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 is sensitive to varying levels of methylation.</i></figcaption></figure></div><br />
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</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 />
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</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 />
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<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 />
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<b><center><h1>Background Information</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<div align="left"><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>Epigenetics.</b></h4> 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 />
</br><br />
<h4><b>DNA Methylation.</b></h4> DNA methylation is one of the most prominent and powerful mechanisms of epigenetic control. In mammals, enzymes called methyltransferases catalyze the addition of methyl groups to cytosines in CpG sites (a cytosine next to a guanine); these sites are abundant throughout the human genome (Venter 2001). Methylated cytosines in CpG sites can block transcription factors from binding to DNA and repress gene expression (Jones 1998 and Nan 1998). Although epigenetic factors do not affect genotype, they do affect phenotype. Specific methylation patterns are necessary for healthy development and disruption of methylation patterns has been shown to lead to many diseases (Cooper 1988, Rideout III 1990, Baylin 1998, Jones 1999, and Amir 1999).<br />
</br><br />
</br><br />
</br><br />
<b><center><h1>An Unmet Need</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Engineering.</b></h4> Synthetic biology largely involved engineering genetic networks in bacterial chasses at its inception, but there have been increasing efforts to engineer more complex mammalian systems. Yet, despite the dramatic effects that subtle epigenetic modifications can have on phenotype, there are no robust and well-characterized tools for engineering the epigenome. If synthetic biologists intend to successfully transition from bacterial to mammalian chasses, they must appreciate the epigenetic control of gene expression. An epigenetic toolbox for synthetic biology would enable the creation of engineered organisms that more closely rival their natural counterparts with regard to the subtlety and robustness of cellular control. We decided to make one of these tools, an enzyme that can direct DNA methylation to specific sequences.<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/b2/Transcriptional-Silencing.png" alt="In Vivo Methylation" width="600"><figcaption><i>Figure 1: Methylation-mediated transcriptional silencing could grant synthetic biologists access to an additional layer of genetic control.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Disease.</b></h4> Methylation abnormalities are linked to a wide range of diseases. Many types of cancer can be characterized by their DNA methylation profiles. In fact, DNA methylation has been called “the hallmark of cancer” (Syzf, 2004). Specifically, hypomethylation of oncogenes has been linked to tumorigenesis and loss of CpG methylation at specific sites has been implicated as a main cause of cancer. No drugs, approved or in testing, can restore methylation. We determined to engineer enzymes that are up to the task.<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/2013/7/78/Healthy-to-Cancer.png" alt="Hypomethylation" width="600"><figcaption><i>Figure 2: Site-specific methylation could potentially restore normal methylation levels to hypomethylated cells.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<b><center><h1>Existing Technologies</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Zinc-Finger Systems.</b> </h4>Some progress has been made towards developing a tool that can make methylate DNA in a controlled manner (Xu 1997, Carvin 2003, and van Steensel 2000). Methylases are the enzymes which catalyze DNA methylation, but they are not inherently targeted to any specific DNA sequence. Since the 1990s, zinc finger proteins that bind a given DNA target sequence have been fused to methylases in an attempt to create an enzyme capable of methylating predetermined DNA sequences (Xu 1997). Although these fusion proteins have been somewhat successful in directing and controlling DNA methylation, they are known to methylate “off-target” DNA sequences distinct from the region intended to be methylated, it is difficult to modify the zinc finger domain to target unique DNA sequences, and the protein engineering process is expensive (Li 2006, Papwort 2005, and Desjarlais 1992). For these reasons, zinc finger methyltransferase fusion proteins have not gained wide spread use in epigenetic studies, and have not been considered for therapeutic purposes. We recapitulated the results with published zinc finger fusions, but were eager to improve on these existing technologies.<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/2013/2/2d/Msssi.gif" alt="M.SssI" width="600" height="500"><figcaption><i>Molecular model of M.SssI (Generated using Phyre2)</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>TAL Effector Systems.</b></h4> The TALE is a more site specific and easily customizable DNA-binding domain than the zinc finger, which has increasingly been used in synthetic biology DNA targeting experiments. As we worked on our project this summer and fall, we were thrilled to see other labs thinking about epigenetic engineering. In the past few months, there has been published work on TALEs fused with histone methylases, histone demethylases, and DNA demethylases (Konermann 2013, Mendenhall 2013, Maedner 2013). The contribution of our TALE DNA methylase completes this initial suite of tools for epigenetic engineering.<br />
</br><br />
</br><br />
</br><br />
<h4><b>Slowed Progress.</b></h4>DNA binding proteins that are more modular and specific to their target sequence than zinc-fingers, such as the TALE and CRISPR-Cas systems, have gained widespread attention for their utility in genetic studies, but they have not been leveraged for improved targeted methylation. Screening new methyltransferase fusions for activity and specificity is difficult and expensive, which could hamper protein-engineering efforts (Gaj 2013). So, we developed a new methylation assay, MaGellin, to accelerate development and cut costs. Then, we used MaGellin to prove the efficacy of the first TALE-methylase fusion.<br />
</br><br />
</br><br />
<h4><b>Noise Problem.</b></h4>An additional challenge that makes the design and characterization of proteins which methylate specific DNA sequences is the signal:noise ratio in mammalian cells. With a large amount of CpG sites naturally methylated in mammalian cells, it is difficult to differentiate protein activity from background noise. However, CpG methylation is completely orthogonal to E.coli so we developed our methylation assay, MaGellin, in this bacterial chassis.<br />
</br><br />
</br><br />
<h4><b>Lack of a Standarized Assay.</b></h4>The most pressing challenge, however, was the lack of a standardized assay for the activity of site-specific methylases that was inexpensive, fast, robust, and easy to use. So, the first element of our project and our most important contribution to the community was the development of the MaGellin assay.<br />
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<b><center><h1>Background Information</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<div align="left"><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>Epigenetics.</b></h4> 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 />
</br><br />
<h4><b>DNA Methylation.</b></h4> DNA methylation is one of the most prominent and powerful mechanisms of epigenetic control. In mammals, enzymes called methyltransferases catalyze the addition of methyl groups to cytosines in CpG sites (a cytosine next to a guanine); these sites are abundant throughout the human genome (Venter 2001). Methylated cytosines in CpG sites can block transcription factors from binding to DNA and repress gene expression (Jones 1998 and Nan 1998). Although epigenetic factors do not affect genotype, they do affect phenotype. Specific methylation patterns are necessary for healthy development and disruption of methylation patterns has been shown to lead to many diseases (Cooper 1988, Rideout III 1990, Baylin 1998, Jones 1999, and Amir 1999).<br />
</br><br />
</br><br />
</br><br />
<b><center><h1>An Unmet Need</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Engineering.</b></h4> Synthetic biology largely involved engineering genetic networks in bacterial chasses at its inception, but there have been increasing efforts to engineer more complex mammalian systems. Yet, despite the dramatic effects that subtle epigenetic modifications can have on phenotype, there are no robust and well-characterized tools for engineering the epigenome. If synthetic biologists intend to successfully transition from bacterial to mammalian chasses, they must appreciate the epigenetic control of gene expression. An epigenetic toolbox for synthetic biology would enable the creation of engineered organisms that more closely rival their natural counterparts with regard to the subtlety and robustness of cellular control. We decided to make one of these tools, an enzyme that can direct DNA methylation to specific sequences.<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/b2/Transcriptional-Silencing.png" alt="In Vivo Methylation" width="600"><figcaption><i>Figure 1: Methylation-mediated transcriptional silencing would allow synthetic biologists access to an additional layer of genetic control.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Disease.</b></h4> Methylation abnormalities are linked to a wide range of diseases. Many types of cancer can be characterized by their DNA methylation profiles. In fact, DNA methylation has been called “the hallmark of cancer” (Syzf, 2004). Specifically, hypomethylation of oncogenes has been linked to tumorigenesis and loss of CpG methylation at specific sites has been implicated as a main cause of cancer. No drugs, approved or in testing, can restore methylation. We determined to engineer enzymes that are up to the task.<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/2013/7/78/Healthy-to-Cancer.png" alt="Hypomethylation" width="600"><figcaption><i>SFigure 2: Site-specific methylation could potentially restore normal methylation levels to hypomethylated cells.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<b><center><h1>Existing Technologies</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Zinc-Finger Systems.</b> </h4>Some progress has been made towards developing a tool that can make methylate DNA in a controlled manner (Xu 1997, Carvin 2003, and van Steensel 2000). Methylases are the enzymes which catalyze DNA methylation, but they are not inherently targeted to any specific DNA sequence. Since the 1990s, zinc finger proteins that bind a given DNA target sequence have been fused to methylases in an attempt to create an enzyme capable of methylating predetermined DNA sequences (Xu 1997). Although these fusion proteins have been somewhat successful in directing and controlling DNA methylation, they are known to methylate “off-target” DNA sequences distinct from the region intended to be methylated, it is difficult to modify the zinc finger domain to target unique DNA sequences, and the protein engineering process is expensive (Li 2006, Papwort 2005, and Desjarlais 1992). For these reasons, zinc finger methyltransferase fusion proteins have not gained wide spread use in epigenetic studies, and have not been considered for therapeutic purposes. We recapitulated the results with published zinc finger fusions, but were eager to improve on these existing technologies.<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/2013/2/2d/Msssi.gif" alt="M.SssI" width="600" height="500"><figcaption><i>Molecular model of M.SssI (Generated using Phyre2)</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>TAL Effector Systems.</b></h4> The TALE is a more site specific and easily customizable DNA-binding domain than the zinc finger, which has increasingly been used in synthetic biology DNA targeting experiments. As we worked on our project this summer and fall, we were thrilled to see other labs thinking about epigenetic engineering. In the past few months, there has been published work on TALEs fused with histone methylases, histone demethylases, and DNA demethylases (Konermann 2013, Mendenhall 2013, Maedner 2013). The contribution of our TALE DNA methylase completes this initial suite of tools for epigenetic engineering.<br />
</br><br />
</br><br />
</br><br />
<h4><b>Slowed Progress.</b></h4>DNA binding proteins that are more modular and specific to their target sequence than zinc-fingers, such as the TALE and CRISPR-Cas systems, have gained widespread attention for their utility in genetic studies, but they have not been leveraged for improved targeted methylation. Screening new methyltransferase fusions for activity and specificity is difficult and expensive, which could hamper protein-engineering efforts (Gaj 2013). So, we developed a new methylation assay, MaGellin, to accelerate development and cut costs. Then, we used MaGellin to prove the efficacy of the first TALE-methylase fusion.<br />
</br><br />
</br><br />
<h4><b>Noise Problem.</b></h4>An additional challenge that makes the design and characterization of proteins which methylate specific DNA sequences is the signal:noise ratio in mammalian cells. With a large amount of CpG sites naturally methylated in mammalian cells, it is difficult to differentiate protein activity from background noise. However, CpG methylation is completely orthogonal to E.coli so we developed our methylation assay, MaGellin, in this bacterial chassis.<br />
</br><br />
</br><br />
<h4><b>Lack of a Standarized Assay.</b></h4>The most pressing challenge, however, was the lack of a standardized assay for the activity of site-specific methylases that was inexpensive, fast, robust, and easy to use. So, the first element of our project and our most important contribution to the community was the development of the MaGellin assay.<br />
</br></br><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 />
<i>Click on the image below to play a step-by-step animation that details the process.</i><br />
<center><object data="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf" type="application/x-shockwave-flash" width="600px" height = "400px"><br />
<br />
<param name="movie" value="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf"><br />
</object><br />
<figcaption><i>Interactive animation of MaGellin workflow</i></figcaption><br />
</center><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>Figure 2: 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 />
<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 3: The full workflow to use MaGellin, available from the BioBrick registry.</i></figcaption></figure></div><br />
</br><br />
</br><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 />
<i>Click on the image below to play a step-by-step animation that details the process.</i><br />
<center><object data="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf" type="application/x-shockwave-flash" width="600px" height = "400px"><br />
<br />
<param name="movie" value="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf"><br />
</object><br />
<figcaption><i>Interactive animation of MaGellin workflow</i></figcaption><br />
</center><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>Figure 2: 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 />
<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"></br><figcaption><i>Figure 3: The full workflow to use MaGellin, available from the BioBrick registry.</i></figcaption></figure></div><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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</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 />
<i>Click on the image below to play a step-by-step animation that details the process.</i><br />
<center><object data="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf" type="application/x-shockwave-flash" width="600px" height = "400px"><br />
<br />
<param name="movie" value="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf"><br />
</object><br />
<figcaption><i>Interactive animation of MaGellin workflow</i></figcaption><br />
</center><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>Figure 2: 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 />
<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 3: The full workflow to use MaGellin, available from the BioBrick registry.</i></figcaption></figure></div><br />
</br><br />
</br><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 />
<i>Click on the image below to play a step-by-step animation that details the process.</i><br />
<center><object data="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf" type="application/x-shockwave-flash" width="600px" height = "400px"><br />
<br />
<param name="movie" value="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf"><br />
</object><br />
<figcaption><i>Interactive animation of MaGellin workflow</i></figcaption><br />
</center><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 />
<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 />
<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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</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 />
<i>Click on the image below to start the animation.</i><br />
<center><object data="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf" type="application/x-shockwave-flash" width="600px" height = "400px"><br />
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<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 />
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 />
<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 />
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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 />
<i>Click on the image below to start the animation.</i><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 />
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 />
<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 />
<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 />
<i>Click on the image below to start the animation.</i><br />
<center><object data="https://dl.dropboxusercontent.com/u/105935696/2013_iGEM_FinalAnimation%20(1).swf" type="application/x-shockwave-flash" width="600px"><br />
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<figcaption><i>Figure 3: Interactive animation of MaGellin workflow</i></figcaption><br />
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</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 />
<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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<b><center><h1>Background Information</h1></center></b><br />
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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.<br />
</br><br />
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<h4><b>Epigenetics.</b></h4> 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 />
</br><br />
<h4><b>DNA Methylation.</b></h4> DNA methylation is one of the most prominent and powerful mechanisms of epigenetic control. In mammals, enzymes called methyltransferases catalyze the addition of methyl groups to cytosines in CpG sites (a cytosine next to a guanine); these sites are abundant throughout the human genome (Venter 2001). Methylated cytosines in CpG sites can block transcription factors from binding to DNA and repress gene expression (Jones 1998 and Nan 1998). Although epigenetic factors do not affect genotype, they do affect phenotype. Specific methylation patterns are necessary for healthy development and disruption of methylation patterns has been shown to lead to many diseases (Cooper 1988, Rideout III 1990, Baylin 1998, Jones 1999, and Amir 1999).<br />
</br><br />
</br><br />
</br><br />
<b><center><h1>An Unmet Need</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Engineering.</b></h4> Synthetic biology largely involved engineering genetic networks in bacterial chasses at its inception, but there have been increasing efforts to engineer more complex mammalian systems. Yet, despite the dramatic effects that subtle epigenetic modifications can have on phenotype, there are no robust and well-characterized tools for engineering the epigenome. If synthetic biologists intend to successfully transition from bacterial to mammalian chasses, they must appreciate the epigenetic control of gene expression. An epigenetic toolbox for synthetic biology would enable the creation of engineered organisms that more closely rival their natural counterparts with regard to the subtlety and robustness of cellular control. We decided to make one of these tools, an enzyme that can direct DNA methylation to specific sequences.<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/b2/Transcriptional-Silencing.png" alt="In Vivo Methylation" width="600"><figcaption><i>Methylation-mediated transcriptional silencing would allow synthetic biologists access to an additional layer of genetic control.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Disease.</b></h4> Methylation abnormalities are linked to a wide range of diseases. Many types of cancer can be characterized by their DNA methylation profiles. In fact, DNA methylation has been called “the hallmark of cancer” (Syzf, 2004). Specifically, hypomethylation of oncogenes has been linked to tumorigenesis and loss of CpG methylation at specific sites has been implicated as a main cause of cancer. No drugs, approved or in testing, can restore methylation. We determined to engineer enzymes that are up to the task.<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/2013/7/78/Healthy-to-Cancer.png" alt="Hypomethylation" width="600"><figcaption><i>Site-specific methylation could potentially restore normal methylation levels to hypomethylated cells.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<b><center><h1>Existing Technologies</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Zinc-Finger Systems.</b> </h4>Some progress has been made towards developing a tool that can make methylate DNA in a controlled manner (Xu 1997, Carvin 2003, and van Steensel 2000). Methylases are the enzymes which catalyze DNA methylation, but they are not inherently targeted to any specific DNA sequence. Since the 1990s, zinc finger proteins that bind a given DNA target sequence have been fused to methylases in an attempt to create an enzyme capable of methylating predetermined DNA sequences (Xu 1997). Although these fusion proteins have been somewhat successful in directing and controlling DNA methylation, they are known to methylate “off-target” DNA sequences distinct from the region intended to be methylated, it is difficult to modify the zinc finger domain to target unique DNA sequences, and the protein engineering process is expensive (Li 2006, Papwort 2005, and Desjarlais 1992). For these reasons, zinc finger methyltransferase fusion proteins have not gained wide spread use in epigenetic studies, and have not been considered for therapeutic purposes. We recapitulated the results with published zinc finger fusions, but were eager to improve on these existing technologies.<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/2013/2/2d/Msssi.gif" alt="M.SssI" width="600" height="500"><figcaption><i>Molecular model of M.SssI (Generated using Phyre2)</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>TAL Effector Systems.</b></h4> The TALE is a more site specific and easily customizable DNA-binding domain than the zinc finger, which has increasingly been used in synthetic biology DNA targeting experiments. As we worked on our project this summer and fall, we were thrilled to see other labs thinking about epigenetic engineering. In the past few months, there has been published work on TALEs fused with histone methylases, histone demethylases, and DNA demethylases (Konermann 2013, Mendenhall 2013, Maedner 2013). The contribution of our TALE DNA methylase completes this initial suite of tools for epigenetic engineering.<br />
</br><br />
</br><br />
</br><br />
<h4><b>Slowed Progress.</b></h4>DNA binding proteins that are more modular and specific to their target sequence than zinc-fingers, such as the TALE and CRISPR-Cas systems, have gained widespread attention for their utility in genetic studies, but they have not been leveraged for improved targeted methylation. Screening new methyltransferase fusions for activity and specificity is difficult and expensive, which could hamper protein-engineering efforts (Gaj 2013). So, we developed a new methylation assay, MaGellin, to accelerate development and cut costs. Then, we used MaGellin to prove the efficacy of the first TALE-methylase fusion.<br />
</br><br />
</br><br />
<h4><b>Noise Problem.</b></h4>An additional challenge that makes the design and characterization of proteins which methylate specific DNA sequences is the signal:noise ratio in mammalian cells. With a large amount of CpG sites naturally methylated in mammalian cells, it is difficult to differentiate protein activity from background noise. However, CpG methylation is completely orthogonal to E.coli so we developed our methylation assay, MaGellin, in this bacterial chassis.<br />
</br><br />
</br><br />
<h4><b>Lack of a Standarized Assay.</b></h4>The most pressing challenge, however, was the lack of a standardized assay for the activity of site-specific methylases that was inexpensive, fast, robust, and easy to use. So, the first element of our project and our most important contribution to the community was the development of the MaGellin assay.<br />
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<b><center><h1>Background Information</h1></center></b><br />
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</br><br />
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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.<br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetics.</b></h4> 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 />
</br><br />
<h4><b>DNA Methylation.</b></h4> DNA methylation is one of the most prominent and powerful mechanisms of epigenetic control. In mammals, enzymes called methyltransferases catalyze the addition of methyl groups to cytosines in CpG sites (a cytosine next to a guanine); these sites are abundant throughout the human genome (Venter 2001). Methylated cytosines in CpG sites can block transcription factors from binding to DNA and repress gene expression (Jones 1998 and Nan 1998). Although epigenetic factors do not affect genotype, they do affect phenotype. Specific methylation patterns are necessary for healthy development and disruption of methylation patterns has been shown to lead to many diseases (Cooper 1988, Rideout III 1990, Baylin 1998, Jones 1999, and Amir 1999).<br />
</br><br />
</br><br />
</br><br />
<b><center><h1>An Unmet Need</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Engineering.</b></h4> Synthetic biology largely involved engineering genetic networks in bacterial chasses at its inception, but there have been increasing efforts to engineer more complex mammalian systems. Yet, despite the dramatic effects that subtle epigenetic modifications can have on phenotype, there are no robust and well-characterized tools for engineering the epigenome. If synthetic biologists intend to successfully transition from bacterial to mammalian chasses, they must appreciate the epigenetic control of gene expression. An epigenetic toolbox for synthetic biology would enable the creation of engineered organisms that more closely rival their natural counterparts with regard to the subtlety and robustness of cellular control. We decided to make one of these tools, an enzyme that can direct DNA methylation to specific sequences.<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/b2/Transcriptional-Silencing.png" alt="In Vivo Methylation" width="600"><figcaption><i>In Vivo Methylation</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Disease.</b></h4> Methylation abnormalities are linked to a wide range of diseases. Many types of cancer can be characterized by their DNA methylation profiles. In fact, DNA methylation has been called “the hallmark of cancer” (Syzf, 2004). Specifically, hypomethylation of oncogenes has been linked to tumorigenesis and loss of CpG methylation at specific sites has been implicated as a main cause of cancer. No drugs, approved or in testing, can restore methylation. We determined to engineer enzymes that are up to the task.<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/2013/7/78/Healthy-to-Cancer.png" alt="Hypomethylation" width="600"><figcaption><i>Hypomethylation can lead to cancer.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<b><center><h1>Existing Technologies</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Zinc-Finger Systems.</b> </h4>Some progress has been made towards developing a tool that can make methylate DNA in a controlled manner (Xu 1997, Carvin 2003, and van Steensel 2000). Methylases are the enzymes which catalyze DNA methylation, but they are not inherently targeted to any specific DNA sequence. Since the 1990s, zinc finger proteins that bind a given DNA target sequence have been fused to methylases in an attempt to create an enzyme capable of methylating predetermined DNA sequences (Xu 1997). Although these fusion proteins have been somewhat successful in directing and controlling DNA methylation, they are known to methylate “off-target” DNA sequences distinct from the region intended to be methylated, it is difficult to modify the zinc finger domain to target unique DNA sequences, and the protein engineering process is expensive (Li 2006, Papwort 2005, and Desjarlais 1992). For these reasons, zinc finger methyltransferase fusion proteins have not gained wide spread use in epigenetic studies, and have not been considered for therapeutic purposes. We recapitulated the results with published zinc finger fusions, but were eager to improve on these existing technologies.<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/2013/2/2d/Msssi.gif" alt="M.SssI" width="600" height="500"><figcaption><i>Molecular model of M.SssI (Generated using Phyre2)</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>TAL Effector Systems.</b></h4> The TALE is a more site specific and easily customizable DNA-binding domain than the zinc finger, which has increasingly been used in synthetic biology DNA targeting experiments. As we worked on our project this summer and fall, we were thrilled to see other labs thinking about epigenetic engineering. In the past few months, there has been published work on TALEs fused with histone methylases, histone demethylases, and DNA demethylases (Konermann 2013, Mendenhall 2013, Maedner 2013). The contribution of our TALE DNA methylase completes this initial suite of tools for epigenetic engineering.<br />
</br><br />
</br><br />
</br><br />
<h4><b>Slowed Progress.</b></h4>DNA binding proteins that are more modular and specific to their target sequence than zinc-fingers, such as the TALE and CRISPR-Cas systems, have gained widespread attention for their utility in genetic studies, but they have not been leveraged for improved targeted methylation. Screening new methyltransferase fusions for activity and specificity is difficult and expensive, which could hamper protein-engineering efforts (Gaj 2013). So, we developed a new methylation assay, MaGellin, to accelerate development and cut costs. Then, we used MaGellin to prove the efficacy of the first TALE-methylase fusion.<br />
</br><br />
</br><br />
<h4><b>Noise Problem.</b></h4>An additional challenge that makes the design and characterization of proteins which methylate specific DNA sequences is the signal:noise ratio in mammalian cells. With a large amount of CpG sites naturally methylated in mammalian cells, it is difficult to differentiate protein activity from background noise. However, CpG methylation is completely orthogonal to E.coli so we developed our methylation assay, MaGellin, in this bacterial chassis.<br />
</br><br />
</br><br />
<h4><b>Lack of a Standarized Assay.</b></h4>The most pressing challenge, however, was the lack of a standardized assay for the activity of site-specific methylases that was inexpensive, fast, robust, and easy to use. So, the first element of our project and our most important contribution to the community was the development of the MaGellin assay.<br />
</br></br><br />
<br />
<br />
<br />
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<b><center><h1>Background Information</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<div align="left"><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>Epigenetics.</b></h4> 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 />
</br><br />
<h4><b>DNA Methylation.</b></h4> DNA methylation is one of the most prominent and powerful mechanisms of epigenetic control. In mammals, enzymes called methyltransferases catalyze the addition of methyl groups to cytosines in CpG sites (a cytosine next to a guanine); these sites are abundant throughout the human genome (Venter 2001). Methylated cytosines in CpG sites can block transcription factors from binding to DNA and repress gene expression (Jones 1998 and Nan 1998). Although epigenetic factors do not affect genotype, they do affect phenotype. Specific methylation patterns are necessary for healthy development and disruption of methylation patterns has been shown to lead to many diseases (Cooper 1988, Rideout III 1990, Baylin 1998, Jones 1999, and Amir 1999).<br />
</br><br />
</br><br />
</br><br />
<b><center><h1>An Unmet Need</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Engineering.</b></h4> Synthetic biology largely involved engineering genetic networks in bacterial chasses at its inception, but there have been increasing efforts to engineer more complex mammalian systems. Yet, despite the dramatic effects that subtle epigenetic modifications can have on phenotype, there are no robust and well-characterized tools for engineering the epigenome. If synthetic biologists intend to successfully transition from bacterial to mammalian chasses, they must appreciate the epigenetic control of gene expression. An epigenetic toolbox for synthetic biology would enable the creation of engineered organisms that more closely rival their natural counterparts with regard to the subtlety and robustness of cellular control. We decided to make one of these tools, an enzyme that can direct DNA methylation to specific sequences.<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/2013/8/8d/In_Vivo_Methylation.jpg" alt="In Vivo Methylation" width="400" height="200"><figcaption><i>In Vivo Methylation</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Disease.</b></h4> Methylation abnormalities are linked to a wide range of diseases. Many types of cancer can be characterized by their DNA methylation profiles. In fact, DNA methylation has been called “the hallmark of cancer” (Syzf, 2004). Specifically, hypomethylation of oncogenes has been linked to tumorigenesis and loss of CpG methylation at specific sites has been implicated as a main cause of cancer. No drugs, approved or in testing, can restore methylation. We determined to engineer enzymes that are up to the task.<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/2013/7/78/Healthy-to-Cancer.png" alt="Hypomethylation" width="600"><figcaption><i>Hypomethylation can lead to cancer.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<b><center><h1>Existing Technologies</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Zinc-Finger Systems.</b> </h4>Some progress has been made towards developing a tool that can make methylate DNA in a controlled manner (Xu 1997, Carvin 2003, and van Steensel 2000). Methylases are the enzymes which catalyze DNA methylation, but they are not inherently targeted to any specific DNA sequence. Since the 1990s, zinc finger proteins that bind a given DNA target sequence have been fused to methylases in an attempt to create an enzyme capable of methylating predetermined DNA sequences (Xu 1997). Although these fusion proteins have been somewhat successful in directing and controlling DNA methylation, they are known to methylate “off-target” DNA sequences distinct from the region intended to be methylated, it is difficult to modify the zinc finger domain to target unique DNA sequences, and the protein engineering process is expensive (Li 2006, Papwort 2005, and Desjarlais 1992). For these reasons, zinc finger methyltransferase fusion proteins have not gained wide spread use in epigenetic studies, and have not been considered for therapeutic purposes. We recapitulated the results with published zinc finger fusions, but were eager to improve on these existing technologies.<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/2013/2/2d/Msssi.gif" alt="M.SssI" width="600" height="500"><figcaption><i>Molecular model of M.SssI (Generated using Phyre2)</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>TAL Effector Systems.</b></h4> The TALE is a more site specific and easily customizable DNA-binding domain than the zinc finger, which has increasingly been used in synthetic biology DNA targeting experiments. As we worked on our project this summer and fall, we were thrilled to see other labs thinking about epigenetic engineering. In the past few months, there has been published work on TALEs fused with histone methylases, histone demethylases, and DNA demethylases (Konermann 2013, Mendenhall 2013, Maedner 2013). The contribution of our TALE DNA methylase completes this initial suite of tools for epigenetic engineering.<br />
</br><br />
</br><br />
</br><br />
<h4><b>Slowed Progress.</b></h4>DNA binding proteins that are more modular and specific to their target sequence than zinc-fingers, such as the TALE and CRISPR-Cas systems, have gained widespread attention for their utility in genetic studies, but they have not been leveraged for improved targeted methylation. Screening new methyltransferase fusions for activity and specificity is difficult and expensive, which could hamper protein-engineering efforts (Gaj 2013). So, we developed a new methylation assay, MaGellin, to accelerate development and cut costs. Then, we used MaGellin to prove the efficacy of the first TALE-methylase fusion.<br />
</br><br />
</br><br />
<h4><b>Noise Problem.</b></h4>An additional challenge that makes the design and characterization of proteins which methylate specific DNA sequences is the signal:noise ratio in mammalian cells. With a large amount of CpG sites naturally methylated in mammalian cells, it is difficult to differentiate protein activity from background noise. However, CpG methylation is completely orthogonal to E.coli so we developed our methylation assay, MaGellin, in this bacterial chassis.<br />
</br><br />
</br><br />
<h4><b>Lack of a Standarized Assay.</b></h4>The most pressing challenge, however, was the lack of a standardized assay for the activity of site-specific methylases that was inexpensive, fast, robust, and easy to use. So, the first element of our project and our most important contribution to the community was the development of the MaGellin assay.<br />
</br></br><br />
<br />
<br />
<br />
<br />
<center><a href="https://2013.igem.org/Team:Penn/MaGellinToolbox">Next&#8594;</a></center><br />
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<b><center><h1>Background Information</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<div align="left"><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>Epigenetics.</b></h4> 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 />
</br><br />
<h4><b>DNA Methylation.</b></h4> DNA methylation is one of the most prominent and powerful mechanisms of epigenetic control. In mammals, enzymes called methyltransferases catalyze the addition of methyl groups to cytosines in CpG sites (a cytosine next to a guanine); these sites are abundant throughout the human genome (Venter 2001). Methylated cytosines in CpG sites can block transcription factors from binding to DNA and repress gene expression (Jones 1998 and Nan 1998). Although epigenetic factors do not affect genotype, they do affect phenotype. Specific methylation patterns are necessary for healthy development and disruption of methylation patterns has been shown to lead to many diseases (Cooper 1988, Rideout III 1990, Baylin 1998, Jones 1999, and Amir 1999).<br />
</br><br />
</br><br />
</br><br />
<b><center><h1>An Unmet Need</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Engineering.</b></h4> Synthetic biology largely involved engineering genetic networks in bacterial chasses at its inception, but there have been increasing efforts to engineer more complex mammalian systems. Yet, despite the dramatic effects that subtle epigenetic modifications can have on phenotype, there are no robust and well-characterized tools for engineering the epigenome. If synthetic biologists intend to successfully transition from bacterial to mammalian chasses, they must appreciate the epigenetic control of gene expression. An epigenetic toolbox for synthetic biology would enable the creation of engineered organisms that more closely rival their natural counterparts with regard to the subtlety and robustness of cellular control. We decided to make one of these tools, an enzyme that can direct DNA methylation to specific sequences.<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/2013/8/8d/In_Vivo_Methylation.jpg" alt="In Vivo Methylation" width="400" height="200"><figcaption><i>In Vivo Methylation</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Disease.</b></h4> Methylation abnormalities are linked to a wide range of diseases. Many types of cancer can be characterized by their DNA methylation profiles. In fact, DNA methylation has been called “the hallmark of cancer” (Syzf, 2004). Specifically, hypomethylation of oncogenes has been linked to tumorigenesis and loss of CpG methylation at specific sites has been implicated as a main cause of cancer. No drugs, approved or in testing, can restore methylation. We determined to engineer enzymes that are up to the task.<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/2013/7/78/Healthy-to-Cancer.png" alt="Hypomethylation" width="600" height="400"><figcaption><i>Hypomethylation can lead to cancer.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<b><center><h1>Existing Technologies</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Zinc-Finger Systems.</b> </h4>Some progress has been made towards developing a tool that can make methylate DNA in a controlled manner (Xu 1997, Carvin 2003, and van Steensel 2000). Methylases are the enzymes which catalyze DNA methylation, but they are not inherently targeted to any specific DNA sequence. Since the 1990s, zinc finger proteins that bind a given DNA target sequence have been fused to methylases in an attempt to create an enzyme capable of methylating predetermined DNA sequences (Xu 1997). Although these fusion proteins have been somewhat successful in directing and controlling DNA methylation, they are known to methylate “off-target” DNA sequences distinct from the region intended to be methylated, it is difficult to modify the zinc finger domain to target unique DNA sequences, and the protein engineering process is expensive (Li 2006, Papwort 2005, and Desjarlais 1992). For these reasons, zinc finger methyltransferase fusion proteins have not gained wide spread use in epigenetic studies, and have not been considered for therapeutic purposes. We recapitulated the results with published zinc finger fusions, but were eager to improve on these existing technologies.<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/2013/2/2d/Msssi.gif" alt="M.SssI" width="600" height="500"><figcaption><i>Molecular model of M.SssI (Generated using Phyre2)</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>TAL Effector Systems.</b></h4> The TALE is a more site specific and easily customizable DNA-binding domain than the zinc finger, which has increasingly been used in synthetic biology DNA targeting experiments. As we worked on our project this summer and fall, we were thrilled to see other labs thinking about epigenetic engineering. In the past few months, there has been published work on TALEs fused with histone methylases, histone demethylases, and DNA demethylases (Konermann 2013, Mendenhall 2013, Maedner 2013). The contribution of our TALE DNA methylase completes this initial suite of tools for epigenetic engineering.<br />
</br><br />
</br><br />
</br><br />
<h4><b>Slowed Progress.</b></h4>DNA binding proteins that are more modular and specific to their target sequence than zinc-fingers, such as the TALE and CRISPR-Cas systems, have gained widespread attention for their utility in genetic studies, but they have not been leveraged for improved targeted methylation. Screening new methyltransferase fusions for activity and specificity is difficult and expensive, which could hamper protein-engineering efforts (Gaj 2013). So, we developed a new methylation assay, MaGellin, to accelerate development and cut costs. Then, we used MaGellin to prove the efficacy of the first TALE-methylase fusion.<br />
</br><br />
</br><br />
<h4><b>Noise Problem.</b></h4>An additional challenge that makes the design and characterization of proteins which methylate specific DNA sequences is the signal:noise ratio in mammalian cells. With a large amount of CpG sites naturally methylated in mammalian cells, it is difficult to differentiate protein activity from background noise. However, CpG methylation is completely orthogonal to E.coli so we developed our methylation assay, MaGellin, in this bacterial chassis.<br />
</br><br />
</br><br />
<h4><b>Lack of a Standarized Assay.</b></h4>The most pressing challenge, however, was the lack of a standardized assay for the activity of site-specific methylases that was inexpensive, fast, robust, and easy to use. So, the first element of our project and our most important contribution to the community was the development of the MaGellin assay.<br />
</br></br><br />
<br />
<br />
<br />
<br />
<center><a href="https://2013.igem.org/Team:Penn/MaGellinToolbox">Next&#8594;</a></center><br />
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<b><center><h1>Background Information</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<div align="left"><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>Epigenetics.</b></h4> 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 />
</br><br />
<h4><b>DNA Methylation.</b></h4> DNA methylation is one of the most prominent and powerful mechanisms of epigenetic control. In mammals, enzymes called methyltransferases catalyze the addition of methyl groups to cytosines in CpG sites (a cytosine next to a guanine); these sites are abundant throughout the human genome (Venter 2001). Methylated cytosines in CpG sites can block transcription factors from binding to DNA and repress gene expression (Jones 1998 and Nan 1998). Although epigenetic factors do not affect genotype, they do affect phenotype. Specific methylation patterns are necessary for healthy development and disruption of methylation patterns has been shown to lead to many diseases (Cooper 1988, Rideout III 1990, Baylin 1998, Jones 1999, and Amir 1999).<br />
</br><br />
</br><br />
</br><br />
<b><center><h1>An Unmet Need</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Engineering.</b></h4> Synthetic biology largely involved engineering genetic networks in bacterial chasses at its inception, but there have been increasing efforts to engineer more complex mammalian systems. Yet, despite the dramatic effects that subtle epigenetic modifications can have on phenotype, there are no robust and well-characterized tools for engineering the epigenome. If synthetic biologists intend to successfully transition from bacterial to mammalian chasses, they must appreciate the epigenetic control of gene expression. An epigenetic toolbox for synthetic biology would enable the creation of engineered organisms that more closely rival their natural counterparts with regard to the subtlety and robustness of cellular control. We decided to make one of these tools, an enzyme that can direct DNA methylation to specific sequences.<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/2013/8/8d/In_Vivo_Methylation.jpg" alt="In Vivo Methylation" width="400" height="200"><figcaption><i>In Vivo Methylation</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Disease.</b></h4> Methylation abnormalities are linked to a wide range of diseases. Many types of cancer can be characterized by their DNA methylation profiles. In fact, DNA methylation has been called “the hallmark of cancer” (Syzf, 2004). Specifically, hypomethylation of oncogenes has been linked to tumorigenesis and loss of CpG methylation at specific sites has been implicated as a main cause of cancer. No drugs, approved or in testing, can restore methylation. We determined to engineer enzymes that are up to the task.<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/2013/7/78/Healthy-to-Cancer.png" alt="Hypomethylation" width="400" height="200"><figcaption><i>Hypomethylation can lead to cancer.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<b><center><h1>Existing Technologies</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Zinc-Finger Systems.</b> </h4>Some progress has been made towards developing a tool that can make methylate DNA in a controlled manner (Xu 1997, Carvin 2003, and van Steensel 2000). Methylases are the enzymes which catalyze DNA methylation, but they are not inherently targeted to any specific DNA sequence. Since the 1990s, zinc finger proteins that bind a given DNA target sequence have been fused to methylases in an attempt to create an enzyme capable of methylating predetermined DNA sequences (Xu 1997). Although these fusion proteins have been somewhat successful in directing and controlling DNA methylation, they are known to methylate “off-target” DNA sequences distinct from the region intended to be methylated, it is difficult to modify the zinc finger domain to target unique DNA sequences, and the protein engineering process is expensive (Li 2006, Papwort 2005, and Desjarlais 1992). For these reasons, zinc finger methyltransferase fusion proteins have not gained wide spread use in epigenetic studies, and have not been considered for therapeutic purposes. We recapitulated the results with published zinc finger fusions, but were eager to improve on these existing technologies.<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/2013/2/2d/Msssi.gif" alt="M.SssI" width="600" height="500"><figcaption><i>Molecular model of M.SssI (Generated using Phyre2)</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>TAL Effector Systems.</b></h4> The TALE is a more site specific and easily customizable DNA-binding domain than the zinc finger, which has increasingly been used in synthetic biology DNA targeting experiments. As we worked on our project this summer and fall, we were thrilled to see other labs thinking about epigenetic engineering. In the past few months, there has been published work on TALEs fused with histone methylases, histone demethylases, and DNA demethylases (Konermann 2013, Mendenhall 2013, Maedner 2013). The contribution of our TALE DNA methylase completes this initial suite of tools for epigenetic engineering.<br />
</br><br />
</br><br />
</br><br />
<h4><b>Slowed Progress.</b></h4>DNA binding proteins that are more modular and specific to their target sequence than zinc-fingers, such as the TALE and CRISPR-Cas systems, have gained widespread attention for their utility in genetic studies, but they have not been leveraged for improved targeted methylation. Screening new methyltransferase fusions for activity and specificity is difficult and expensive, which could hamper protein-engineering efforts (Gaj 2013). So, we developed a new methylation assay, MaGellin, to accelerate development and cut costs. Then, we used MaGellin to prove the efficacy of the first TALE-methylase fusion.<br />
</br><br />
</br><br />
<h4><b>Noise Problem.</b></h4>An additional challenge that makes the design and characterization of proteins which methylate specific DNA sequences is the signal:noise ratio in mammalian cells. With a large amount of CpG sites naturally methylated in mammalian cells, it is difficult to differentiate protein activity from background noise. However, CpG methylation is completely orthogonal to E.coli so we developed our methylation assay, MaGellin, in this bacterial chassis.<br />
</br><br />
</br><br />
<h4><b>Lack of a Standarized Assay.</b></h4>The most pressing challenge, however, was the lack of a standardized assay for the activity of site-specific methylases that was inexpensive, fast, robust, and easy to use. So, the first element of our project and our most important contribution to the community was the development of the MaGellin assay.<br />
</br></br><br />
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<b><center><h1>Background Information</h1></center></b><br />
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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.<br />
</br><br />
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<h4><b>Epigenetics.</b></h4> 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 />
</br><br />
<h4><b>DNA Methylation.</b></h4> DNA methylation is one of the most prominent and powerful mechanisms of epigenetic control. In mammals, enzymes called methyltransferases catalyze the addition of methyl groups to cytosines in CpG sites (a cytosine next to a guanine); these sites are abundant throughout the human genome (Venter 2001). Methylated cytosines in CpG sites can block transcription factors from binding to DNA and repress gene expression (Jones 1998 and Nan 1998). Although epigenetic factors do not affect genotype, they do affect phenotype. Specific methylation patterns are necessary for healthy development and disruption of methylation patterns has been shown to lead to many diseases (Cooper 1988, Rideout III 1990, Baylin 1998, Jones 1999, and Amir 1999).<br />
</br><br />
</br><br />
</br><br />
<b><center><h1>An Unmet Need</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Engineering.</b></h4> Synthetic biology largely involved engineering genetic networks in bacterial chasses at its inception, but there have been increasing efforts to engineer more complex mammalian systems. Yet, despite the dramatic effects that subtle epigenetic modifications can have on phenotype, there are no robust and well-characterized tools for engineering the epigenome. If synthetic biologists intend to successfully transition from bacterial to mammalian chasses, they must appreciate the epigenetic control of gene expression. An epigenetic toolbox for synthetic biology would enable the creation of engineered organisms that more closely rival their natural counterparts with regard to the subtlety and robustness of cellular control. We decided to make one of these tools, an enzyme that can direct DNA methylation to specific sequences.<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/2013/8/8d/In_Vivo_Methylation.jpg" alt="In Vivo Methylation" width="400" height="200"><figcaption><i>In Vivo Methylation</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>Epigenetic Disease.</b></h4> Methylation abnormalities are linked to a wide range of diseases. Many types of cancer can be characterized by their DNA methylation profiles. In fact, DNA methylation has been called “the hallmark of cancer” (Syzf, 2004). Specifically, hypomethylation of oncogenes has been linked to tumorigenesis and loss of CpG methylation at specific sites has been implicated as a main cause of cancer. No drugs, approved or in testing, can restore methylation. We determined to engineer enzymes that are up to the task.<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/2013/4/47/Hypomethylation.jpg" alt="Hypomethylation" width="400" height="200"><figcaption><i>Hypomethylation can lead to cancer.</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<b><center><h1>Existing Technologies</h1></center></b><br />
</br><br />
</br><br />
</br><br />
<h4><b>Zinc-Finger Systems.</b> </h4>Some progress has been made towards developing a tool that can make methylate DNA in a controlled manner (Xu 1997, Carvin 2003, and van Steensel 2000). Methylases are the enzymes which catalyze DNA methylation, but they are not inherently targeted to any specific DNA sequence. Since the 1990s, zinc finger proteins that bind a given DNA target sequence have been fused to methylases in an attempt to create an enzyme capable of methylating predetermined DNA sequences (Xu 1997). Although these fusion proteins have been somewhat successful in directing and controlling DNA methylation, they are known to methylate “off-target” DNA sequences distinct from the region intended to be methylated, it is difficult to modify the zinc finger domain to target unique DNA sequences, and the protein engineering process is expensive (Li 2006, Papwort 2005, and Desjarlais 1992). For these reasons, zinc finger methyltransferase fusion proteins have not gained wide spread use in epigenetic studies, and have not been considered for therapeutic purposes. We recapitulated the results with published zinc finger fusions, but were eager to improve on these existing technologies.<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/2013/2/2d/Msssi.gif" alt="M.SssI" width="600" height="500"><figcaption><i>Molecular model of M.SssI (Generated using Phyre2)</i></figcaption></figure></div><br />
</br><br />
</br><br />
</br><br />
<h4><b>TAL Effector Systems.</b></h4> The TALE is a more site specific and easily customizable DNA-binding domain than the zinc finger, which has increasingly been used in synthetic biology DNA targeting experiments. As we worked on our project this summer and fall, we were thrilled to see other labs thinking about epigenetic engineering. In the past few months, there has been published work on TALEs fused with histone methylases, histone demethylases, and DNA demethylases (Konermann 2013, Mendenhall 2013, Maedner 2013). The contribution of our TALE DNA methylase completes this initial suite of tools for epigenetic engineering.<br />
</br><br />
</br><br />
</br><br />
<h4><b>Slowed Progress.</b></h4>DNA binding proteins that are more modular and specific to their target sequence than zinc-fingers, such as the TALE and CRISPR-Cas systems, have gained widespread attention for their utility in genetic studies, but they have not been leveraged for improved targeted methylation. Screening new methyltransferase fusions for activity and specificity is difficult and expensive, which could hamper protein-engineering efforts (Gaj 2013). So, we developed a new methylation assay, MaGellin, to accelerate development and cut costs. Then, we used MaGellin to prove the efficacy of the first TALE-methylase fusion.<br />
</br><br />
</br><br />
<h4><b>Noise Problem.</b></h4>An additional challenge that makes the design and characterization of proteins which methylate specific DNA sequences is the signal:noise ratio in mammalian cells. With a large amount of CpG sites naturally methylated in mammalian cells, it is difficult to differentiate protein activity from background noise. However, CpG methylation is completely orthogonal to E.coli so we developed our methylation assay, MaGellin, in this bacterial chassis.<br />
</br><br />
</br><br />
<h4><b>Lack of a Standarized Assay.</b></h4>The most pressing challenge, however, was the lack of a standardized assay for the activity of site-specific methylases that was inexpensive, fast, robust, and easy to use. So, the first element of our project and our most important contribution to the community was the development of the MaGellin assay.<br />
</br></br><br />
<br />
<br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
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Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
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</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 />
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</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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<center><br />
<embed src = "https://sites.google.com/site/pennigemhosting/2013_iGEM_FinalAnimation.swf" height = "600" width = "800"><br />
</center><br />
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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<embed src = "https://sites.google.com/site/pennigemhosting/2013_iGEM_FinalAnimation.swf"><br />
<br />
<>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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
</html></div>Magaracihttp://2013.igem.org/Team:Penn/AssayOverviewTeam:Penn/AssayOverview2013-10-28T06:54:40Z<p>Magaraci: </p>
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<embed src = "https://sites.google.com/site/pennigemhosting/2013_iGEM_FinalAnimation.swf"></embed><br />
<br />
<>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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<embed src = "https://swiffypreviews.googleusercontent.com/view/o/9e04d263-21c1-4e49-bff0-af44c660434c/2013_iGEM_FinalAnimation.html""></embed><br />
<br />
<>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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<iframe src = "https://swiffypreviews.googleusercontent.com/view/o/9e04d263-21c1-4e49-bff0-af44c660434c/2013_iGEM_FinalAnimation.html""></iframe><br />
<br />
<>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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<br />
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<br />
<>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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
</html></div>Magaracihttp://2013.igem.org/Team:Penn/AssayOverviewTeam:Penn/AssayOverview2013-10-28T05:49:50Z<p>Magaraci: </p>
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> 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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
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<br />
<br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
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<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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
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classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000"<br />
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</embed><br />
</object><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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<OBJECT classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=6,0,0,0" WIDTH="800" HEIGHT="600" id="Yourfilename" ALIGN=""><br />
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<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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<OBJECT classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=6,0,0,0" WIDTH="800" HEIGHT="600" id="Yourfilename" ALIGN=""><br />
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<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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
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<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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> 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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<br />
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<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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
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<br />
<br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<br />
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<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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
</html></div>Magaracihttp://2013.igem.org/Team:Penn/AssayOverviewTeam:Penn/AssayOverview2013-10-28T05:23:19Z<p>Magaraci: </p>
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
</div><br />
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<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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
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<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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
</html></div>Magaracihttp://2013.igem.org/Team:Penn/AssayOverviewTeam:Penn/AssayOverview2013-10-28T02:09:27Z<p>Magaraci: </p>
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<object src = "https://sites.google.com/site/pennigemhosting/2013_iGEM_FinalAnimation.swf" classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" width="800" height="600" id="FlashID" title="animation"><br />
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<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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
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<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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
</div><br />
</div><br />
<br />
<br />
</body><br />
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<header><h1><b><center>Assay Overview</center></b></h1></header><br />
</br><br />
</br><br />
</br><br />
Early on, it became clear that there was no standardized assay for site-specific methylases. Importantly, it seemed many groups were not focusing enough on their methylases tendency to also methylate off-target sequences; and were only measuring the site-specific methylation.<br />
</br><br />
</br><br />
<b>Measuring Methylation</b> 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 unmethylated cytosines converted 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”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, 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 accomodate screening libraries of candidate DNA-binding-domain-methyltransferase 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 />
<b>Our Team’s Solution.</b> 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 methyltransferase, 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 – methyltransferase 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/2013/9/9d/Plasmid-Schematic-Updated.png" alt="MaGellin" width="700" height="395"><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 />
<b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<br />
<ol><br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must 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 CRISPR-Cas, TALE, Zinc Finger, 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 fortify MaGellin’s results with bisulfite sequencing results</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>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
</br><br />
</br><br />
</br><br />
<b>Noiseless Chassis.</b> 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 />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><br />
<br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" width="800" height="600" id="FlashID" title="animation" src = "https://dl.dropboxusercontent.com/u/17048378/2013_iGEM_FinalAnimation.swf"><br />
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<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 />
<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg" 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 />
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</html></div>Magaracihttp://2013.igem.org/File:Avin-Zinc-Graph.pngFile:Avin-Zinc-Graph.png2013-09-28T03:51:54Z<p>Magaraci: </p>
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<div></div>Magaracihttp://2013.igem.org/File:Avin-Tale-Graph-for-Mike.pngFile:Avin-Tale-Graph-for-Mike.png2013-09-28T03:48:43Z<p>Magaraci: </p>
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<div></div>Magaracihttp://2013.igem.org/Team:Penn/MaGellinResultsTeam:Penn/MaGellinResults2013-09-28T03:46:16Z<p>Magaraci: </p>
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<b><center><h1><br />
MaGellin Results<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://dl.dropboxusercontent.com/u/11828463/MaGellin%20Spec%20Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
<br><br />
<br><br />
<center><br />
<div align=left><br />
<b>Our Team’s Solution</b><br />
In order to address the challenges associated with developing new site-specific methyltransferase 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 methyltransferase, 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 – methyltransferase fusion protein. This plasmid-based methylation assay is called MaGellin.<br><br />
<br><b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<ol><br />
<br />
<br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must spend cloning. </li><br />
<br />
<li>Multiple cloning site downstream of T7 promoter for orthogonal expression of fusion protein in T7 Express competent E. coli.</li><br />
<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 CRISPR-Cas, TALE, Zinc Finger, or transcription factor<br />
</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 />
<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 />
<br />
<li>XbaI site for linearization of the plasmid. Linearizing the plasmid simplifies analysis of the AvaI digestion by gel electrophoresis.</li><br />
<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 fortify MaGellin’s results with bisulfite sequencing results</li><br />
<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 />
<br />
<li>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/f/fb/Bisulfite_vs.png" width=600><br><br><br />
<i>Figure 1: Bisulfite Sequencing compared to the MaGellin Assay</i><br />
</center><br />
<br />
<br />
<br><br><br />
<b>Noiseless Chassis. </b>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:<ol><br />
<br />
<li>T7 RNA Polymerase in the lac operon allows us to turn on expression of fusion protein after induction with IPTG</li><br />
<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></ol><br />
<br />
<br />
<br />
<br><br><br />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><ol><br />
<b>Assemble</b> the MaGellin backbone together with a DNA-binding protein and target sequence of your choosing.<ol><br />
<li>Digest BBa_K1128001 (the MaGellin backbone) and BBa_K1128002 (the linker-M.ssI construct) with EcoRI and PstI.</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 type="disc"> Forward: CAGGAGGAATTC[ATG] (add start codon only if not included in gene).</li><br />
<li type="disc"> 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> </ol><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.<ol><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 type="disc">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 />
<br />
</ol><br><br><br />
<br />
<br />
<br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/d/d9/Workflow-schematics.png"><br><br></center><br />
<br />
<center><i>Figure 2: An illustration depicting the typical workflow for MaGellin.</i><br><br></center><br />
<br />
<b>Complementary Software. </b>We realized early on that the MaGellin assay could easily lend itself to quantification, and we designed a software package to do just that. The MaGellin software accelerates experimental analysis and removes human bias. It is unique because its bioinformatics module can predict expected band lengths based on the methylation sensitivity of the restriction enzymes.<br><br><br />
<br />
<center><b>Validating MaGellin</b></center><br />
<b>With Methyltransferase.</b> First, we tested MaGellin with a purified methyltransferase in vitro. The results made it clear that MaGellin can detect methylation, at both the “target” and “off-target” site. MaGellin is also sensitive to various degrees of methylation. These experiments helped us optimize the ideal amount of plasmid and restriction enzyme to use in any study moving forward.<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/1/16/Time-Course-Data.png"><br><br><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/InVitroValidation.jpg" width=600><br><br></center><br />
<br />
<b>With a Validated Fusion.</b> Now that we knew MaGellin could report DNA methylation in a quantifiable manner, we wanted to express an older fusion. We recreated the old zinc finger – methyltransferase fusion protein that had been previously described and assayed its activity and specificity. What we found using MaGellin was in agreement with the numerous studies that had focused on assaying this fusion protein. We found that overexpression of the zinc finger fusion protein led to many off target methylation events, confirming what others had noted about this fusion protein – that it is not suitable for site specific DNA methylation (Figure 3).<br />
<br />
<br />
<br><center><br />
<img src="https://static.igem.org/mediawiki/2013/6/6b/Zinc_Finger_GEl.jpg" width="600"><br><br><br />
<i>Figure 3: Expression of a previously published zinc finger-methyltransferase fusion in E.coli. We observed significant off-target effects.</i></center><br><br />
<br><br />
<b>Summary</b><br><ol><br />
<li>We have created MaGellin, a new technology that facilitates screening novel DNA binding domain – methyltransferase fusion proteins</li><br />
<br />
<li>Our assay is less expensive and faster than existing methods</li><br />
<br />
<li>We have eliminated noise associated with previous studies</li><br />
<br />
<li>We have a system with clear input/output</li><br />
<br />
<li>Our assay lends itself to high throughput screening of many different proteins</li><br />
<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 />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
</html></div>Magaracihttp://2013.igem.org/Team:Penn/MaGellinResultsTeam:Penn/MaGellinResults2013-09-28T03:45:39Z<p>Magaraci: </p>
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<div class="section1" style="background-position: top;"><br />
<div class="text"><br />
<b><center><h1><br />
MaGellin Results<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://dl.dropboxusercontent.com/u/11828463/MaGellin%20Spec%20Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
<br><br />
<br><br />
<center><br />
<div align=left><br />
<b>Our Team’s Solution</b><br />
In order to address the challenges associated with developing new site-specific methyltransferase 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 methyltransferase, 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 – methyltransferase fusion protein. This plasmid-based methylation assay is called MaGellin.<br><br />
<br><b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<ol><br />
<br />
<br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must spend cloning. </li><br />
<br />
<li>Multiple cloning site downstream of T7 promoter for orthogonal expression of fusion protein in T7 Express competent E. coli.</li><br />
<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 CRISPR-Cas, TALE, Zinc Finger, or transcription factor<br />
</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 />
<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 />
<br />
<li>XbaI site for linearization of the plasmid. Linearizing the plasmid simplifies analysis of the AvaI digestion by gel electrophoresis.</li><br />
<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 fortify MaGellin’s results with bisulfite sequencing results</li><br />
<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 />
<br />
<li>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/f/fb/Bisulfite_vs.png" width=600><br><br><br />
<i>Figure 1: Bisulfite Sequencing compared to the MaGellin Assay</i><br />
</center><br />
<br />
<br />
<br><br><br />
<b>Noiseless Chassis. </b>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:<ol><br />
<br />
<li>T7 RNA Polymerase in the lac operon allows us to turn on expression of fusion protein after induction with IPTG</li><br />
<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></ol><br />
<br />
<br />
<br />
<br><br><br />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><ol><br />
<b>Assemble</b> the MaGellin backbone together with a DNA-binding protein and target sequence of your choosing.<ol><br />
<li>Digest BBa_K1128001 (the MaGellin backbone) and BBa_K1128002 (the linker-M.ssI construct) with EcoRI and PstI.</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 type="disc"> Forward: CAGGAGGAATTC[ATG] (add start codon only if not included in gene).</li><br />
<li type="disc"> 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> </ol><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.<ol><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 type="disc">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 />
<br />
</ol><br><br><br />
<br />
<br />
<br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/d/d9/Workflow-schematics.png"><br><br></center><br />
<br />
<center><i>Figure 2: An illustration depicting the typical workflow for MaGellin.</i><br><br></center><br />
<br />
<b>Complementary Software. </b>We realized early on that the MaGellin assay could easily lend itself to quantification, and we designed a software package to do just that. The MaGellin software accelerates experimental analysis and removes human bias. It is unique because its bioinformatics module can predict expected band lengths based on the methylation sensitivity of the restriction enzymes.<br><br><br />
<br />
<center><b>Validating MaGellin</b></center><br />
<b>With Methyltransferase.</b> First, we tested MaGellin with a purified methyltransferase in vitro. The results made it clear that MaGellin can detect methylation, at both the “target” and “off-target” site. MaGellin is also sensitive to various degrees of methylation. These experiments helped us optimize the ideal amount of plasmid and restriction enzyme to use in any study moving forward.<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/1/16/Time-Course-Data.png"><br><br></center><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/InVitroValidation.jpg" width=600><br><br></center><br />
<br />
<b>With a Validated Fusion.</b> Now that we knew MaGellin could report DNA methylation in a quantifiable manner, we wanted to express an older fusion. We recreated the old zinc finger – methyltransferase fusion protein that had been previously described and assayed its activity and specificity. What we found using MaGellin was in agreement with the numerous studies that had focused on assaying this fusion protein. We found that overexpression of the zinc finger fusion protein led to many off target methylation events, confirming what others had noted about this fusion protein – that it is not suitable for site specific DNA methylation (Figure 3).<br />
<br />
<br />
<br><center><br />
<img src="https://static.igem.org/mediawiki/2013/6/6b/Zinc_Finger_GEl.jpg" width="600"><br><br><br />
<i>Figure 3: Expression of a previously published zinc finger-methyltransferase fusion in E.coli. We observed significant off-target effects.</i></center><br><br />
<br><br />
<b>Summary</b><br><ol><br />
<li>We have created MaGellin, a new technology that facilitates screening novel DNA binding domain – methyltransferase fusion proteins</li><br />
<br />
<li>Our assay is less expensive and faster than existing methods</li><br />
<br />
<li>We have eliminated noise associated with previous studies</li><br />
<br />
<li>We have a system with clear input/output</li><br />
<br />
<li>Our assay lends itself to high throughput screening of many different proteins</li><br />
<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 />
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<b><center><h1><br />
MaGellin Results<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://dl.dropboxusercontent.com/u/11828463/MaGellin%20Spec%20Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
<br><br />
<br><br />
<center><br />
<div align=left><br />
<b>Our Team’s Solution</b><br />
In order to address the challenges associated with developing new site-specific methyltransferase 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 methyltransferase, 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 – methyltransferase fusion protein. This plasmid-based methylation assay is called MaGellin.<br><br />
<br><b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<ol><br />
<br />
<br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must spend cloning. </li><br />
<br />
<li>Multiple cloning site downstream of T7 promoter for orthogonal expression of fusion protein in T7 Express competent E. coli.</li><br />
<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 CRISPR-Cas, TALE, Zinc Finger, or transcription factor<br />
</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 />
<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 />
<br />
<li>XbaI site for linearization of the plasmid. Linearizing the plasmid simplifies analysis of the AvaI digestion by gel electrophoresis.</li><br />
<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 fortify MaGellin’s results with bisulfite sequencing results</li><br />
<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 />
<br />
<li>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/f/fb/Bisulfite_vs.png" width=600><br><br><br />
<i>Figure 1: Bisulfite Sequencing compared to the MaGellin Assay</i><br />
</center><br />
<br />
<br />
<br><br><br />
<b>Noiseless Chassis. </b>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:<ol><br />
<br />
<li>T7 RNA Polymerase in the lac operon allows us to turn on expression of fusion protein after induction with IPTG</li><br />
<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></ol><br />
<br />
<br />
<br />
<br><br><br />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><ol><br />
<b>Assemble</b> the MaGellin backbone together with a DNA-binding protein and target sequence of your choosing.<ol><br />
<li>Digest BBa_K1128001 (the MaGellin backbone) and BBa_K1128002 (the linker-M.ssI construct) with EcoRI and PstI.</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 type="disc"> Forward: CAGGAGGAATTC[ATG] (add start codon only if not included in gene).</li><br />
<li type="disc"> 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> </ol><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.<ol><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 type="disc">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 />
<br />
</ol><br><br><br />
<br />
<br />
<br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/d/d9/Workflow-schematics.png"><br><br></center><br />
<br />
<center><i>Figure 2: An illustration depicting the typical workflow for MaGellin.</i><br><br></center><br />
<br />
<b>Complementary Software. </b>We realized early on that the MaGellin assay could easily lend itself to quantification, and we designed a software package to do just that. The MaGellin software accelerates experimental analysis and removes human bias. It is unique because its bioinformatics module can predict expected band lengths based on the methylation sensitivity of the restriction enzymes.<br><br><br />
<br />
<center><b>Validating MaGellin</b></center><br />
<b>With Methyltransferase.</b> First, we tested MaGellin with a purified methyltransferase in vitro. The results made it clear that MaGellin can detect methylation, at both the “target” and “off-target” site. MaGellin is also sensitive to various degrees of methylation. These experiments helped us optimize the ideal amount of plasmid and restriction enzyme to use in any study moving forward.<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/InVitroValidation.jpg" width=600><br><br></center><br />
<br />
<b>With a Validated Fusion.</b> Now that we knew MaGellin could report DNA methylation in a quantifiable manner, we wanted to express an older fusion. We recreated the old zinc finger – methyltransferase fusion protein that had been previously described and assayed its activity and specificity. What we found using MaGellin was in agreement with the numerous studies that had focused on assaying this fusion protein. We found that overexpression of the zinc finger fusion protein led to many off target methylation events, confirming what others had noted about this fusion protein – that it is not suitable for site specific DNA methylation (Figure 3).<br />
<br />
<br />
<br><center><br />
<img src="https://static.igem.org/mediawiki/2013/6/6b/Zinc_Finger_GEl.jpg" width="600"><br><br><br />
<i>Figure 3: Expression of a previously published zinc finger-methyltransferase fusion in E.coli. We observed significant off-target effects.</i></center><br><br />
<br><br />
<b>Summary</b><br><ol><br />
<li>We have created MaGellin, a new technology that facilitates screening novel DNA binding domain – methyltransferase fusion proteins</li><br />
<br />
<li>Our assay is less expensive and faster than existing methods</li><br />
<br />
<li>We have eliminated noise associated with previous studies</li><br />
<br />
<li>We have a system with clear input/output</li><br />
<br />
<li>Our assay lends itself to high throughput screening of many different proteins</li><br />
<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 />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
</html></div>Magaracihttp://2013.igem.org/Team:Penn/MaGellinResultsTeam:Penn/MaGellinResults2013-09-28T03:43:12Z<p>Magaraci: </p>
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<div class="section1" style="background-position: top;"><br />
<div class="text"><br />
<b><center><h1><br />
MaGellin Results<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://dl.dropboxusercontent.com/u/11828463/MaGellin%20Spec%20Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
<br><br />
<br><br />
<center><br />
<div align=left><br />
<b>Our Team’s Solution</b><br />
In order to address the challenges associated with developing new site-specific methyltransferase 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 methyltransferase, 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 – methyltransferase fusion protein. This plasmid-based methylation assay is called MaGellin.<br><br />
<br><b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<ol><br />
<br />
<br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must spend cloning. </li><br />
<br />
<li>Multiple cloning site downstream of T7 promoter for orthogonal expression of fusion protein in T7 Express competent E. coli.</li><br />
<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 CRISPR-Cas, TALE, Zinc Finger, or transcription factor<br />
</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 />
<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 />
<br />
<li>XbaI site for linearization of the plasmid. Linearizing the plasmid simplifies analysis of the AvaI digestion by gel electrophoresis.</li><br />
<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 fortify MaGellin’s results with bisulfite sequencing results</li><br />
<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 />
<br />
<li>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/f/fb/Bisulfite_vs.png" width=600><br><br><br />
<i>Figure 1: Bisulfite Sequencing compared to the MaGellin Assay</i><br />
</center><br />
<br />
<br />
<br><br><br />
<b>Noiseless Chassis. </b>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:<ol><br />
<br />
<li>T7 RNA Polymerase in the lac operon allows us to turn on expression of fusion protein after induction with IPTG</li><br />
<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></ol><br />
<br />
<br />
<br />
<br><br><br />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><ol><br />
<b>Assemble</b> the MaGellin backbone together with a DNA-binding protein and target sequence of your choosing.<ol><br />
<li>Digest BBa_K1128001 (the MaGellin backbone) and BBa_K1128002 (the linker-M.ssI construct) with EcoRI and PstI.</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 type="disc"> Forward: CAGGAGGAATTC[ATG] (add start codon only if not included in gene).</li><br />
<li type="disc"> 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> </ol><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.<ol><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 type="disc">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 />
<br />
</ol><br><br><br />
<br />
<br />
<br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/d/d9/Workflow-schematics.png"><br><br></center><br />
<br />
<center><i>Figure 2: An illustration depicting the typical workflow for MaGellin.</i><br><br></center><br />
<br />
<b>Complementary Software. </b>We realized early on that the MaGellin assay could easily lend itself to quantification, and we designed a software package to do just that. The MaGellin software accelerates experimental analysis and removes human bias. It is unique because its bioinformatics module can predict expected band lengths based on the methylation sensitivity of the restriction enzymes.<br><br><br />
<br />
<center><b>Validating MaGellin</b></center><br />
<b>With Methyltransferase.</b> First, we tested MaGellin with a purified methyltransferase in vitro. The results made it clear that MaGellin can detect methylation, at both the “target” and “off-target” site. MaGellin is also sensitive to various degrees of methylation. These experiments helped us optimize the ideal amount of plasmid and restriction enzyme to use in any study moving forward.<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/1/16/Time-Course-Data.png<br><br></center><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/InVitroValidation.jpg" width=600><br><br></center><br />
<br />
<b>With a Validated Fusion.</b> Now that we knew MaGellin could report DNA methylation in a quantifiable manner, we wanted to express an older fusion. We recreated the old zinc finger – methyltransferase fusion protein that had been previously described and assayed its activity and specificity. What we found using MaGellin was in agreement with the numerous studies that had focused on assaying this fusion protein. We found that overexpression of the zinc finger fusion protein led to many off target methylation events, confirming what others had noted about this fusion protein – that it is not suitable for site specific DNA methylation (Figure 3).<br />
<br />
<br />
<br><center><br />
<img src="https://static.igem.org/mediawiki/2013/6/6b/Zinc_Finger_GEl.jpg" width="600"><br><br><br />
<i>Figure 3: Expression of a previously published zinc finger-methyltransferase fusion in E.coli. We observed significant off-target effects.</i></center><br><br />
<br><br />
<b>Summary</b><br><ol><br />
<li>We have created MaGellin, a new technology that facilitates screening novel DNA binding domain – methyltransferase fusion proteins</li><br />
<br />
<li>Our assay is less expensive and faster than existing methods</li><br />
<br />
<li>We have eliminated noise associated with previous studies</li><br />
<br />
<li>We have a system with clear input/output</li><br />
<br />
<li>Our assay lends itself to high throughput screening of many different proteins</li><br />
<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 />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
</html></div>Magaracihttp://2013.igem.org/Team:Penn/MaGellinResultsTeam:Penn/MaGellinResults2013-09-28T03:42:12Z<p>Magaraci: </p>
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<div class="left_wrap"><br />
</div> </div><br />
<div class="section1" style="background-position: top;"><br />
<div class="text"><br />
<b><center><h1><br />
MaGellin Results<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://dl.dropboxusercontent.com/u/11828463/MaGellin%20Spec%20Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
<br><br />
<br><br />
<center><br />
<div align=left><br />
<b>Our Team’s Solution</b><br />
In order to address the challenges associated with developing new site-specific methyltransferase 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 methyltransferase, 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 – methyltransferase fusion protein. This plasmid-based methylation assay is called MaGellin.<br><br />
<br><b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<ol><br />
<br />
<br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must spend cloning. </li><br />
<br />
<li>Multiple cloning site downstream of T7 promoter for orthogonal expression of fusion protein in T7 Express competent E. coli.</li><br />
<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 CRISPR-Cas, TALE, Zinc Finger, or transcription factor<br />
</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 />
<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 />
<br />
<li>XbaI site for linearization of the plasmid. Linearizing the plasmid simplifies analysis of the AvaI digestion by gel electrophoresis.</li><br />
<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 fortify MaGellin’s results with bisulfite sequencing results</li><br />
<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 />
<br />
<li>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/f/fb/Bisulfite_vs.png" width=600><br><br><br />
<i>Figure 1: Bisulfite Sequencing compared to the MaGellin Assay</i><br />
</center><br />
<br />
<br />
<br><br><br />
<b>Noiseless Chassis. </b>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:<ol><br />
<br />
<li>T7 RNA Polymerase in the lac operon allows us to turn on expression of fusion protein after induction with IPTG</li><br />
<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></ol><br />
<br />
<br />
<br />
<br><br><br />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><ol><br />
<b>Assemble</b> the MaGellin backbone together with a DNA-binding protein and target sequence of your choosing.<ol><br />
<li>Digest BBa_K1128001 (the MaGellin backbone) and BBa_K1128002 (the linker-M.ssI construct) with EcoRI and PstI.</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 type="disc"> Forward: CAGGAGGAATTC[ATG] (add start codon only if not included in gene).</li><br />
<li type="disc"> 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> </ol><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.<ol><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 type="disc">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 />
<br />
</ol><br><br><br />
<br />
<br />
<br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/d/d9/Workflow-schematics.png"><br><br></center><br />
<br />
<center><i>Figure 2: An illustration depicting the typical workflow for MaGellin.</i><br><br></center><br />
<br />
<b>Complementary Software. </b>We realized early on that the MaGellin assay could easily lend itself to quantification, and we designed a software package to do just that. The MaGellin software accelerates experimental analysis and removes human bias. It is unique because its bioinformatics module can predict expected band lengths based on the methylation sensitivity of the restriction enzymes.<br><br><br />
<br />
<center><b>Validating MaGellin</b></center><br />
<b>With Methyltransferase.</b> First, we tested MaGellin with a purified methyltransferase in vitro. The results made it clear that MaGellin can detect methylation, at both the “target” and “off-target” site. MaGellin is also sensitive to various degrees of methylation. These experiments helped us optimize the ideal amount of plasmid and restriction enzyme to use in any study moving forward.<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/1/16/Time-Course-Data.png<br><br></center><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/InVitroValidation.jpg" width=600><br><br></center><br />
<br />
<b>With a Validated Fusion.</b> Now that we knew MaGellin could report DNA methylation in a quantifiable manner, we wanted to express an older fusion. We recreated the old zinc finger – methyltransferase fusion protein that had been previously described and assayed its activity and specificity. What we found using MaGellin was in agreement with the numerous studies that had focused on assaying this fusion protein. We found that overexpression of the zinc finger fusion protein led to many off target methylation events, confirming what others had noted about this fusion protein – that it is not suitable for site specific DNA methylation (Figure 3).<br />
<br />
<br />
<br><center><br />
<img src="https://static.igem.org/mediawiki/2013/6/6b/Zinc_Finger_GEl.jpg" width="600"><br><br><br />
<i>Figure 3: Expression of a previously published zinc finger-methyltransferase fusion in E.coli. We observed significant off-target effects.</i></center><br><br />
<br><br />
<b>Summary</b><br><ol><br />
<li>We have created MaGellin, a new technology that facilitates screening novel DNA binding domain – methyltransferase fusion proteins</li><br />
<br />
<li>Our assay is less expensive and faster than existing methods</li><br />
<br />
<li>We have eliminated noise associated with previous studies</li><br />
<br />
<li>We have a system with clear input/output</li><br />
<br />
<li>Our assay lends itself to high throughput screening of many different proteins</li><br />
<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 />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<br />
</body><br />
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</html></div>Magaracihttp://2013.igem.org/File:Time-Course-Data.pngFile:Time-Course-Data.png2013-09-28T03:40:21Z<p>Magaraci: </p>
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<div></div>Magaracihttp://2013.igem.org/Team:Penn/MaGellinResultsTeam:Penn/MaGellinResults2013-09-28T03:36:25Z<p>Magaraci: </p>
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<b><center><h1><br />
MaGellin Results<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://dl.dropboxusercontent.com/u/11828463/MaGellin%20Spec%20Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow.<br />
<br><br />
<br><br />
<center><br />
<div align=left><br />
<b>Our Team’s Solution</b><br />
In order to address the challenges associated with developing new site-specific methyltransferase 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 methyltransferase, 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 – methyltransferase fusion protein. This plasmid-based methylation assay is called MaGellin.<br><br />
<br><b>Plasmid Features.</b> To accommodate the MaGellin assay, our team designed a plasmid with several key features:<ol><br />
<br />
<br />
<li>CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must spend cloning. </li><br />
<br />
<li>Multiple cloning site downstream of T7 promoter for orthogonal expression of fusion protein in T7 Express competent E. coli.</li><br />
<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 CRISPR-Cas, TALE, Zinc Finger, or transcription factor<br />
</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 />
<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 />
<br />
<li>XbaI site for linearization of the plasmid. Linearizing the plasmid simplifies analysis of the AvaI digestion by gel electrophoresis.</li><br />
<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 fortify MaGellin’s results with bisulfite sequencing results</li><br />
<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 />
<br />
<li>Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.</li><br />
<br />
<li>Kanamycin resistance as a selection marker</li><br />
</ol><br />
<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/f/fb/Bisulfite_vs.png" width=600><br><br><br />
<i>Figure 1: Bisulfite Sequencing compared to the MaGellin Assay</i><br />
</center><br />
<br />
<br />
<br><br><br />
<b>Noiseless Chassis. </b>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:<ol><br />
<br />
<li>T7 RNA Polymerase in the lac operon allows us to turn on expression of fusion protein after induction with IPTG</li><br />
<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></ol><br />
<br />
<br />
<br />
<br><br><br />
<b>MaGellin Workflow.</b> The workflow for screening new fusion proteins with the one plasmid MaGellin bacterial system is as follows:<br><ol><br />
<b>Assemble</b> the MaGellin backbone together with a DNA-binding protein and target sequence of your choosing.<ol><br />
<li>Digest BBa_K1128001 (the MaGellin backbone) and BBa_K1128002 (the linker-M.ssI construct) with EcoRI and PstI.</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 type="disc"> Forward: CAGGAGGAATTC[ATG] (add start codon only if not included in gene).</li><br />
<li type="disc"> 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> </ol><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.<ol><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 type="disc">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 />
<br />
</ol><br><br><br />
<br />
<br />
<br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/d/d9/Workflow-schematics.png"><br><br></center><br />
<br />
<center><i>Figure 2: An illustration depicting the typical workflow for MaGellin.</i><br><br></center><br />
<br />
<b>Complementary Software. </b>We realized early on that the MaGellin assay could easily lend itself to quantification, and we designed a software package to do just that. The MaGellin software accelerates experimental analysis and removes human bias. It is unique because its bioinformatics module can predict expected band lengths based on the methylation sensitivity of the restriction enzymes.<br><br><br />
<br />
<center><b>Validating MaGellin</b></center><br />
<b>With Methyltransferase.</b> First, we tested MaGellin with a purified methyltransferase in vitro. The results made it clear that MaGellin can detect methylation, at both the “target” and “off-target” site. MaGellin is also sensitive to various degrees of methylation. These experiments helped us optimize the ideal amount of plasmid and restriction enzyme to use in any study moving forward.<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/InVitroValidation.jpg" width=600><br><br></center><br />
<br />
<b>With a Validated Fusion.</b> Now that we knew MaGellin could report DNA methylation in a quantifiable manner, we wanted to express an older fusion. We recreated the old zinc finger – methyltransferase fusion protein that had been previously described and assayed its activity and specificity. What we found using MaGellin was in agreement with the numerous studies that had focused on assaying this fusion protein. We found that overexpression of the zinc finger fusion protein led to many off target methylation events, confirming what others had noted about this fusion protein – that it is not suitable for site specific DNA methylation (Figure 3).<br />
<br />
<br />
<br><center><br />
<img src="https://static.igem.org/mediawiki/2013/6/6b/Zinc_Finger_GEl.jpg" width="600"><br><br><br />
<i>Figure 3: Expression of a previously published zinc finger-methyltransferase fusion in E.coli. We observed significant off-target effects.</i></center><br><br />
<br><br />
<b>Summary</b><br><ol><br />
<li>We have created MaGellin, a new technology that facilitates screening novel DNA binding domain – methyltransferase fusion proteins</li><br />
<br />
<li>Our assay is less expensive and faster than existing methods</li><br />
<br />
<li>We have eliminated noise associated with previous studies</li><br />
<br />
<li>We have a system with clear input/output</li><br />
<br />
<li>Our assay lends itself to high throughput screening of many different proteins</li><br />
<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 />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<br />
</body><br />
<script><br />
</script><br />
</html></div>Magaraci