Part:BBa_K1128000
CPG DNA methylase mssSI
Please look at part BBa_K1128001 for more characterization data.
Epigenetics, the study of modifications made to DNA in order to regulate and control the functions of a cell, has several components, one of which is known as DNA methylation. This involves adding a methyl group (-CH3) to a base pair residue, which helps the cell determine if the methylated gene should be off, or if it should be on, and how much protein it should be expressing. The enzymes that catalyze the addition of methyl groups to the DNA are called methyltransferases, and each type of methyltransferase has its own unique activity. The most common type of methylation in a eukaryotic cell is called CpG methylation, which stands for Cytosine-Phosphate-Guanine, and it is the addition of a methyl group to the Cytosine at a CpG site.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 484
Illegal BsaI.rc site found at 365
Illegal SapI site found at 568
| None |