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DNA Engineering - Week 3 APh162 Winter 2007

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Page 1: DNA Engineering - Week 3 APh162 Winter 2007. Quantitative Gene Expression Setty et al. (2003) Small et al. (1992, 1996) Elowitz et al. (2000) How much?

DNA Engineering - Week 3

APh162

Winter 2007

Page 2: DNA Engineering - Week 3 APh162 Winter 2007. Quantitative Gene Expression Setty et al. (2003) Small et al. (1992, 1996) Elowitz et al. (2000) How much?

Quantitative Gene Expression

Setty et al. (2003)

Small et al. (1992, 1996)

Elowitz et al. (2000)

How much? When? Where?

• 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!

Slide courtesy of H. Garcia

Page 3: DNA Engineering - Week 3 APh162 Winter 2007. Quantitative Gene Expression Setty et al. (2003) Small et al. (1992, 1996) Elowitz et al. (2000) How much?

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

Page 4: DNA Engineering - Week 3 APh162 Winter 2007. Quantitative Gene Expression Setty et al. (2003) Small et al. (1992, 1996) Elowitz et al. (2000) How much?

5’ 3’

cAMPRNA Pol

Activator protein

lacZ

5’ 3’

XCAP P Lac rep

5’ 3’

cAMP

Activator protein

XP Lac rep

3’

CAP P O

5’

lacZ Glucose +

Lactose +

Glucose –

Lactose +

Glucose +

Lactose -

Glucose –

Lactose -

The Lac Operon

=>

=>

=>

=>

High expression

Low (leaky)expression

No expression

No expression

[Glucose] 1/[cAMP]

Page 5: DNA Engineering - Week 3 APh162 Winter 2007. Quantitative Gene Expression Setty et al. (2003) Small et al. (1992, 1996) Elowitz et al. (2000) How much?

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!)

IPTG + X-gal

Colonies of cells with the expressed lacZ in their plasmids turn blue!

Page 6: DNA Engineering - Week 3 APh162 Winter 2007. Quantitative Gene Expression Setty et al. (2003) Small et al. (1992, 1996) Elowitz et al. (2000) How much?

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

Page 7: DNA Engineering - Week 3 APh162 Winter 2007. Quantitative Gene Expression Setty et al. (2003) Small et al. (1992, 1996) Elowitz et al. (2000) How much?

LacZ

LacZ LacZ

LacZ

YFP

YFP PCR,purify

Trim,purify

Cut

Purify Ligation

Extract

YFP

Killer cut,purify

KAN

KAN

KANKAN

Transformand plate

KAN

LB + Kanamycin(IPTG, X-Gal)

LacZ

LacZ YFP

Extract and send for sequencing

pZE21-LacZpZS25-YFP

“Vector” “Insert”

Page 8: DNA Engineering - Week 3 APh162 Winter 2007. Quantitative Gene Expression Setty et al. (2003) Small et al. (1992, 1996) Elowitz et al. (2000) How much?

PCR – Polymerase Chain Reaction

Page 9: DNA Engineering - Week 3 APh162 Winter 2007. Quantitative Gene Expression Setty et al. (2003) Small et al. (1992, 1996) Elowitz et al. (2000) How much?

Gel Electrophoresis Ladders

Distance traveled depends on:

• Mass

• Shape

• Electric charge

Graph of the distance traveled vs. mass?


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