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DNA Engineering - Week 3. APh162 Winter 2007. Small et al. (1992, 1996). Elowitz et al. (2000). Setty et al. (2003). Quantitative Gene Expression. Gene expression: Process by which DNA is converted into the structures and functions of the cell

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dna engineering week 3

DNA Engineering - Week 3

APh162

Winter 2007

quantitative gene expression

Small et al. (1992, 1996)

Elowitz et al. (2000)

Setty et al. (2003)

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?

Slide courtesy of H. Garcia

key idea of gene expression the lac operon
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:

Expressed lacZ => -galactosidase => metabolize lactose

the lac operon
The Lac Operon

[Glucose]  1/[cAMP]

Glucose +

Lactose +

P

O

CAP

lacZ

Low (leaky)

expression

=>

5’

3’

Activator protein

RNA Pol

cAMP

Glucose –

Lactose +

High

expression

lacZ

=>

5’

3’

Lac rep

Glucose +

Lactose -

No

expression

P

CAP

=>

X

5’

3’

Activator protein

Glucose –

Lactose -

No

expression

Lac rep

cAMP

=>

P

X

5’

3’

detection of gene expression
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
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

slide7

pZS25-YFP

KAN

KAN

pZE21-LacZ

YFP

Extract

LacZ

Cut

“Vector”

“Insert”

KAN

YFP

PCR,

purify

LacZ

LacZ

Purify

Ligation

Trim,

purify

LacZ

LacZ

YFP

KAN

KAN

Killer cut,

purify

LacZ

YFP

Transform

and plate

Extract and send for sequencing

LB + Kanamycin

(IPTG, X-Gal)

gel electrophoresis
Gel Electrophoresis

Ladders

  • Distance traveled depends on:
  • Mass
  • Shape
  • Electric charge

Graph of the distance traveled vs. mass?