1. DNA Engineering - Week 3
APh162
Winter 2007

2. 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

3. 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

4. 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’

5. 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

6. 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

7. 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)

8. PCR – Polymerase Chain Reaction

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