ChemBiol 474

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Chem/Biol 474 W 2009
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Instructors M/W Gerry Prody CB
444 gerry_at_chem.wwu.edu Scott
Delbec Delbecs_at_cc.wwu.edu T/R Clint
Spiegel CB 443 spiegel_at_wwu.edu Peter
Littlefield
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Course Overview
Lab Notebooks
Prelab/Research Project/Paper
Lab Etiquette
Background Everything in 471 (and 2) is fair
game.
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Figure 5-43 The pUC18 cloning vector.
Page 106
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Figure 5-46 Construction of a recombinant DNA
molecule.
Page 108
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N-terminal 6xHis tag TEV protease
cleavable pET27 derivative T7lac promoter 5
cloning site BamHI low copy plasmid Kan-resistan
t MGSS(H6)ENLYFQ GS-protein ORF
TEVprotease cleavage site
BamHI cloning site
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Figure 31-1 The induction kinetics of
b-galactosidase in E. coli.
Page 1217
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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.

Whats lactose??
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Figure 31-2 Genetic map of the E. coli lac operon.
Page 1218
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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 bacteriums 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.

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Isopropyl thio -? -D- galactoside
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Induction.

• Allolactose is an isomer formed from lactose that
  derepresses the operon by inactivating the
  repressor,
• Thus turning on the enzymes for lactose
  metabolism.

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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).

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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.

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Lac movie
Lac and trp
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• Expression tutorial

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Figure 31-25 The base sequence of the lac
operator.
Page 1239
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Figure 31-37a X-ray structure of the lac
repressor-DNA complex.
Page 1249
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• Lac repressor binding to DNA animation
• http//molvis.sdsc.edu/atlas/morphs/lacrep/index.h
  tm

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http//www.ncbi.nlm.nih.gov/pubmed/ http//www.e
xpasy.ch/ http//www.pdb.org/