Biochemistry 2280A Lecture Notes - Gene Knockout, Agarose Gel Electrophoresis, Plasmid

• School: Western University
• Department: Biochemistry
• Course: Biochemistry 2280A
• Professor: Mel Usselman

Document Summary

Prokaryotic organisms differ in that their genes do not contain introns, and share different promoters, translational sequences as well as post-translational modifications. The issue with introns is largely solved through the use of cdna and the different promoters and translational mechanisms can be easily accommodated by the design of the plasmid you choose in cloning yfg. In expression plasmids, it is necessary that they have a bacterial promoter as well as a ribosome-binding site in front of the integrated cdna of yfg to be expressed. The promoter that is used is usually an inducible promoter whereby the human protein is not expressed until the bacterial cell has reached maturation, so as not to negatively impact the development of the e. coli. Once you"ve induced the protein, the protein is isolated from e. coli, purified (protein homogenization and chromatography) and checked for activity.