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