DNA Engineering - Week 3 APh162 Winter 2007

Quantitative Gene Expression Gene expression: Process by which DNA is converted into the structures and functions of the cell We can now talk about gene expression quantitatively We have quantitative data, we need quantitative models! How much? When? Where? Setty et al. (2003) Elowitz et al. (2000) Small et al. (1992, 1996) Slide courtesy of H. Garcia

Key Idea of Gene Expression – The lac Operon Key idea: Proteins are synthesized only when needed Two food sources for E.coli: Glucose and lactose. Lactose requires the synthesis of 3 proteins in order to be metabolized! When glucose is absent: Expression of the lacZ gene means production of beta-galactosidase, an enzyme which cleaves lactose into 2 monosaccharides, glucose and galactose. But how exactly is this regulated? What controls the expression of the lacZ gene? Expressed lacZ => -galactosidase => metabolize lactose

The Lac Operon [Glucose]  1/[cAMP] CAP P O Glucose + Lactose + lacZ Low (leaky) expression => 5’ 3’ Activator protein cAMP RNA Pol Glucose – Lactose + lacZ High expression => 5’ 3’ Lac rep Glucose + Lactose - CAP P No expression X => cAMP is produced by ATP via the enzyme adenylate cyclase, which is inhibited by the presence of glucose. cAMP-CAP bind the lac promoter (Catabolite Activator Protein) Bind to the CAP site. This activates transcription, perhaps by increasing the affinity of the site for RNA polymerase. When lactose is present, it causes the repressor to leave the operator 5’ 3’ Activator protein Glucose – Lactose - Lac rep No expression cAMP P => X 5’ 3’

Detection of Gene Expression How can we detect the high level of gene expression of the lacZ gene? Plate the cells in plates that Do not have glucose (don’t want the repression) IPTG (like lactose, will pull of the lac repressor from the operator => induces the expression) Substitute lactose with X-gal (turns blue when cleaved!) Colonies of cells with the expressed lacZ in their plasmids turn blue! IPTG + X-gal

Gene Expression Quantitatively Look at gene expression 2 different ways YFP fluorescence lacZ expression At different levels of IPTG Does the amount of expression depend on the reporter? Slide courtesy of H. Garcia

Extract and send for sequencing LB + Kanamycin (IPTG, X-Gal) pZS25-YFP KAN KAN pZE21-LacZ YFP Extract LacZ Cut “Vector” “Insert” YFP KAN PCR, purify LacZ LacZ Purify Ligation Trim, purify LacZ LacZ YFP KAN KAN Circular DNA: The bacteria will replicate, since it has an origin of replication Linear DNA: The bacteria will digest, because it is linear. This is why the killer cut works (the cut in the YFP makes the plasmid linear) Killer cut, purify LacZ YFP Transform and plate Extract and send for sequencing LB + Kanamycin (IPTG, X-Gal)

PCR – Polymerase Chain Reaction

Gel Electrophoresis Ladders Distance traveled depends on: Mass Shape Electric charge Graph of the distance traveled vs. mass?