Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Raoul Martin, Drew Dunham
Date created: 2014-10-08 11:00:00
Date modified: 2015-05-08 01:10:48
MCherry fused to K1519123 for Characterization
| Types | DnaRegion |
| Roles | engineered_region Reporter |
| Sequences | BBa_K1519124_sequence (Version 1) |
Description
By inserting the DNA sequence of a desired protein in our part, one will be able to secret a protein of interest, bind it to a Nickel column, and cleave off the desired protein from the tag, resulting in a purified protein of interest. OsmY, the first gene in the device, has been shown to be a successful secretion tag. When fused to a protein of interest, the OsmY fusion translocates into the periplasm, where it forms disulfide bonds and folds. Through an unknown mechanism, the OsmY fusion is secreted out of the cell, into the growth medium. This secretion removes a significant amount of undesired protein contaminants.The media containing the construct can be run through a nickel column for further purification, utilizing the 6x His-tag incorporated into the construct. Once eluted from the column, addition of the TEV-His protease will cleave OsmY from the protein of interest, eliminating any unwanted effects of the device on the desired protein. Upon a second nickel column purification of the eluate, the TEV-His protease and the cleaved OsmY-His construct will remain on the column while the protein of interest runs off, resulting in an easily purified protein from the supernatant of the growth medium.This construct is identical to K1519123, with an MCherry fusion for characterization
Notes
No design information provided.Source
MG1655 E. coli, Tsien Lab Mcherry| Access | Instance | Definition |
| public public public public public public public | BBa_I712074 BBa_B0034 BBa_K892008 BBa_K243004 BBa_K157011 BBa_J18918 BBa_J06505 | BBa_I712074 BBa_B0034 osmY ShortLinke His TEVsite mCherry |
| Sequence Annotation | Location | Component / Role(s) |
| BBa_I712074 BBa_B0034 BBa_K892008 BBa_K243004 BBa_K157011 BBa_J18918 BBa_J06505 | 1,46 55,66 73,678 687,698 707,724 733,753 760,1482 | BBa_I712074 BBa_B0034 osmY ShortLinke His TEVsite mCherry |