BE.109:Protein engineering/Assessing beta-galactosidase

INTRODUCTION

Cells can be considered miniature machines. They use energy to make things, record events, move around, and modify their environment. They differ from standard, mechanical machines in ways that will be further explored in the Systems Engineering experimental module, but for today consider the remarkable and useful fact that you’re working with a protein-making factory. Under the right conditions, the majority of each cell’s protein output can be the protein you’re most interested in. Though each cell is only microns in size, it’s easy to grow lots of them to yield microgram quantities of protein. Most astonishing of all is the ease with which experimental conditions can be changed to affect the protein output in rational and defined ways. The protein of interest can be turned on or off, modified or not. You did this when you added IPTG with or without an unnatural amino acid to your growth media. Today you will assess the consequences of these manipulations.

The cells you are using carry a plasmid that expresses beta-galactosidase when IPTG is added to the growth media. This expression method was originally described in 1986 (Studier and Moffatt J Mol Biol. Volume 189 (1): 113-130 [[1]]) and then commercialized by a company called Novagen. Two specialized components are used. First is an expression plasmid in which the gene of interest, in this case the LacZ gene that encodes beta-galactosidase, is downstream of a special bacterial promoter. The promoter is not normally active since it’s recognized and used only by an RNA polymerase from the T7 bacteriophage. The gene for the T7 RNA polymerase has been added to the genome of the cells you’re using. Ironically enough, it’s the promoter from the LacZ gene that’s regulating the T7 RNA polymerase expression. Without inducer, the LacZ promoter is off, so there is no T7 RNA polymerase made and so no LacZ is expressed from the plasmid. With IPTG, the lacZ promoter is de-repressed, leading to expression of the T7 RNA polymerase, which transcribes lots of LacZ, and viola…lots of beta-galactosidase. High levels of expression can be expected since the T7 RNA polymerase promoter is strong and each cell maintains many copies of the expression plasmid.