Team:Penn/Biobricks
From 2013.igem.org
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- | For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https:// | + | For a detailed, graphical explanation of the MaGellin work flow, please download the <a href="https://static.igem.org/mediawiki/2013/e/e5/Spec_Sheet.pdf">MaGellin Workflow Specifications Sheet</a>, which includes all of the steps in the MaGellin workflow. |
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<b>MaGellin Plasmid Backbone <a href="http://parts.igem.org/Part:BBa_K1128001">(BBa_K1128001)</a></b> | <b>MaGellin Plasmid Backbone <a href="http://parts.igem.org/Part:BBa_K1128001">(BBa_K1128001)</a></b> |
Revision as of 19:33, 28 October 2013
Parts Submitted
M.SssI is a methyltransferase derived from Spiroplasma, and is a very common protein used in epigenetics studies. It has been shown to work effectively both in vivo and in vitro, and the CpG methylation that M.SssI exhibits is orthogonal to the bacterial form of methylation, which methylates GATC sites in the genome using an enzyme called the DNA Adenine Methyltransferase (dam). In most bacteria, there are certain restriction enzymes native to the genome that digest CpG methylated sites as protection for the cell against foreign DNA. However, if the M.SssI is expressed in the cell in vivo, it will methylate the genome of the cell and cause the genome to be digested, leading to death of the cell. Therefore, when using this part, it should be noted that experiments and cloning involving this part should be done in cells that don’t have this restriction mechanism, called Mcr and Mrr restriction, which can be purchased from vendors such as New England Biolabs. For the Penn iGEM project, the experiments involving this protein were done in T7 Express cells, purchased from New England Biolabs, which are classified as an Mcr- and Mrr- strain. Part of Penn iGEM's project in 2013 was creating novel targeted methyltransferases, which are fusion proteins consisting of a DNA binding domain, such as a TAL effector, linked to a methyltransferse, M.SssI in our case, and testing those proteins for site specific methylation in vivo using the MaGellin assay workflow. We used this part as a positive control for those studies, in order to compare genome wide methylation and site specific methylation using our novel fusion proteins. CpG Methylase M.SssI with Linker (BBa_K1128002) For full characterization see BBa_K1128000. This is an M.SssI methyltransferase protein with a linker on the N terminus. The part is ready for fusion to transcription factors. The linker is composed of a string of glycine residues.