Instructors: M/WGerry ProdyCB 444 gerry@chem.wwu.edu Scott Delbec Delbecs@cc.wwu.edu T/RClint...

Instructors:

M/WGerry Prody CB 444gerry@chem.wwu.edu

Scott DelbecDelbecs@cc.wwu.edu

T/R Clint Spiegel CB 443spiegel@wwu.edu

Peter Littlefield

Course Overview

Lab Notebooks

Prelab/Research Project/Paper

Lab Etiquette

Background: Everything in 471 (and 2) is fair game.

Figure 5-43 The pUC18 cloning vector.P

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Figure 5-37 Restriction sites.P

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Figure 5-46 Construction of a recombinant DNA molecule.

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N-terminal 6xHis tag; TEV protease cleavable

pET27 derivative

T7lac promoter

5’ cloning site: BamHI

low copy plasmid

Kan-resistant

MGSS(H6)ENLYFQ GS-protein ORF

TEVprotease cleavage site

BamHI cloning site

Figure 31-1 The induction kinetics of -galactosidase in E. coli.

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The lac operon• E-coli uses three

enzymes to take up and metabolize lactose.

• The genes that code for these three enzymes are clustered on a single operon – the lac Operon.

What’s lactose??

Figure 31-2 Genetic map of the E. coli lac operon.

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The lac repressor gene• Prior to these three genes is an operator

region that is responsible for turning these genes on and off.

• When there is not lactose, the gene for the lac repressor switches off the operon by binding to the operator region.

• A bacterium’s prime source of food is glucose.

• So if glucose and lactose are around, the bacterium wants to turn off lactose metabolism in favor of glucose metabolism.

Isopropyl thio - -D- galactoside

Induction.• Allolactose is an isomer formed from lactose that

derepresses the operon by inactivating the repressor,

• Thus turning on the enzymes for lactose metabolism.

The lac operon in action.• When lactose is present, it acts as an inducer of the operon (turns it on).• It enters the cell and binds to the Lac repressor, causing a shape change that so

the repressor falls off. • Now the RNA polymerase is free to move along the DNA and RNA can be made

from the three genes. • Lactose can now be metabolized (broken down).

When the inducer (lactose) is removed

• The repressor returns to its original shape and binds to the DNA, so that RNA polymerase can no longer get past the promoter. No RNA and no protein is made.

• Note that RNA polymerase can still bind to the promoter though it is unable to move past it. That means that when the cell is ready to use the operon, RNA polymerase is already there and waiting to begin transcription.

Lac movie

Lac and trp

• Expression tutorial

Figure 31-25 The base sequence of the lac operator.

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Figure 31-37a X-ray structure of the lac repressor-DNA complex.

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• Lac repressor binding to DNA animation

http://molvis.sdsc.edu/atlas/morphs/lacrep/index.htm

http://www.ncbi.nlm.nih.gov/pubmed/

http://www.expasy.ch/

http://www.pdb.org/