dna engineering - week 3
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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DNA Engineering - Week 3
APh162
Winter 2007
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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
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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
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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]
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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!
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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
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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”
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PCR – Polymerase Chain Reaction
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Gel Electrophoresis Ladders
Distance traveled depends on:
• Mass
• Shape
• Electric charge
Graph of the distance traveled vs. mass?