Bacterial Transformation with pGLO Plasmid

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Bacterial Transformation with (pGLO Plasmid)

• Lab 8 Molecular Biology

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Purpose of this Lab

• Learn how to insert a gene into bacteria
• (Heat Shock)
• Analyze how a gene can transform an organism and
  express that gene
• Provide evidence that bacteria can take in
  foreign DNA in the form of a plasmid
• Reinforce the following process
• DNA ? RNA ? Protein ? Trait
• Observe how genes are regulated

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Applications of Genetic Transformation

• Used in many areas of Biotechnology
• Agriculture (pests, frost, drought)
• Bacteria (oil spills)
• Gene therapy (sick cells into healthy cells)
• Medicine (produce insulin hormones)

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Key Terms to Know

• DNA Plasmid
• Bacteria E. coli (strain HB101K-12)
• Growth media LB Broth (Luria Bertani)
• Ampicillin Antibiotic kills bacteria amp
• Arabinose Sugar source for energy carbon
• Heat shock Process that increases permeability
  of the cell membrane to DNA
• GFP Green Fluorescent Protein (w/UV)

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The Genes of Interest

• Ampicillin resistance
• Gene regulation proteins-activate the GFP gene
  when arabinose is present
• GFP Green Fluorescent Protein
• -originally isolated from the jellyfish
• Aequorea victoria

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Chapters 18 19

• Bacteria
• Viruses Operon Systems

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Key Topics and Text Pgs to Review

• Topic Pgs.
• Bacteria Genetic recombination 346-350
• Plasmids Conjugation
• Transformation (Lab 8)
• Transposons 351-352
• Lac Operon System 353-356
• Regulating Gene Expression
• Viruses DNA, RNA (retroviruses) 338-342
• Lytic Lysogenic Cycle 337-339

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Relative size Differences between of Viruses,
Prokaryotes, and Eukaryotes
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Bacterial Reproduction of DNA
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Transformation

• Uptake of foreign DNA from the environment
• What we did in our lab (pGLO plasmid)
• Requires unique cell-surface proteins on the that
  can recognize similar strands of DNA, bind to it,
  and allow uptake.

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Conjugation and the transfer of the F Plasmid
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Transduction
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Detecting Genetic Recombination in Bacteria
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Expected Results

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Introductory Questions

• Briefly explain the differences between
  Transformation, Conjugation, and Transduction.
  How are these three processes the same? (pgs.
  348-349)
• How is an F plasmid different from an R
  plasmid?
• What are transposable elements and what do they
  do?

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Introductory Questions

• Name the two scientists that discovered the Lac
  operon system.
• How are repressible operons different from
  inducible operons? Give an example of each.
• What is the difference between an operator and a
  promoter?
• Name three example of a virus that has DNA as its
  genetic material and three examples of Viruses
  with RNA as its genetic material.
• Briefly explain what a vaccine is and what it
  does.

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Insertion Sequences Transposable Elements

• Always a part of of chromosomal or plasmid DNA
• Sometimes called jumping genes-never detach
• A single gene for coded for transposase
• Inverted sequences are on each side of an
  insertion sequences. Observed in bacteria only.
• See pg. 352
• Specialized plasmids are constructed using these
  sequences.

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Jacob Monod

• Discovered Lac Operon
• Nobel Prize for Discovering Control of Gene
  Expression

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Regulation of a Metabolic Pathway
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Specialized Genes

• Operator "on/off" switch for operon
• Regulator makes repressors to turn off an
  entire operon
• Repressor Binds to operator, turn off gene
  expression
• Inducer Joins with an active repressor,
  inactivates it
• Co-repressor Joins with inactive repressor,
  converts it to active

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OPERON THEORY

• Operon group of structural genes regulated as a
  unit
• Several genes controlled by an operator site

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Operon Complex

• RNA Polymerase must bind to the promoter site and
  continue past the operator site to transcribe mRNA

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INDUCIBLE Operons

• Usually OFF - to turn ON
• INDUCER needs to bind to an active repressor and
  inactivate it
• RNA Polymerase can then bind and transcribe mRNA
• Ex. Lac operon is an inducible operon

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Inactive Repressor-Lactose Present
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Lac Operon Summary

• Beta-Galactosidase can then be made

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Repressible Operons

• Usually ON - to turn OFF
• Co-repressor needs to bind to an inactive
  repressor and activate it
• RNA Polymerase then cannot bind and transcribe
  mRNA
• Ex. trp operon is a repressible operon
  -trancription is usually on
• -inhibited only by tryptophan (corepressor)

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Inactive Repressor-Tryptophan Absent
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Classic Example of Theory

• Splitting of a disaccharide LACTOSE molecule
  within E. coli (Lac Operon)
• TWO molecules needed to bind to promotor site to
  induce transcription of lactose-splitting
  beta-galactosidase
• One molecule complex of cyclic AMP (cAMP)
  cyclic AMP binding protein (CAP)
• One molecule RNA polymerase

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Lac Operon

• Lactose ONLY used when glucose is not present in
  large quantities
• When glucose is present, cAMP levels are low,
  cAMP cannot bind to CAP and initiate enzyme
  production

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Lac Operon

• In absence of glucose, cAMP levels are HIGH,
  binding to CAP can occur
• Beta-Galactosidase is made

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Lac Operon

• RNA polymerase only binds efficiently when
  cAMP-CAP complex is in place
• Lac Operon an INDUCIBLE Operon
• Lactose an INDUCER
• Binds to repressor and inactivates it

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Operons

• Inducible (lac operon)
• lactose metabolism
• lactose not present repressor active
• operon off
• no transcription for lactose
  enzymes
• lactose present repressor inactive operon on
• inducer molecule inactivates protein
  repressor (allolactose)
• transcription is stimulated when inducer binds
  to a regulatory protein

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Lytic Lysogenic Cycles of a Virus(Lysogenichos
t is not destroyed)
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5 Classes of Viruses-Pg. 340
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Examples of Common Viruses

• DNA RNA
• Herpesvirus Ebola
• Poxvirus Infuenza
• Papovirus (warts) HIV
• Measels, Mumps
• Rabies
• West Nile

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HIV Infection (pgs 340-342)
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HIV infection on a White Blood Cell
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Lac Operon Summary

• Beta-Galactosidase can then be made

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Key Concepts for Chapter 19

• Oncogenes Proto-Oncogenes 370-373
• Tumor Supressor Genes
• McClintoks transposons 375-376

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Introductory Questions

• Why are transposons called jumping genes? What
  purpose do the insertion sequences play?
• What is the difference between an oncogene and a
  tumor repressor gene?

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Molecular Biology of Cancer

• Oncogene cancer-causing genes
• Proto-oncogene normal cellular genes
• How?
  1-movement of DNA chromosome fragments that
  have rejoined incorrectly
  2-amplification increases the number
  of copies of proto-oncogenes
  3-proto-oncogene point mutation protein product
  more active or more resistant to degradation
• Tumor-suppressor genes changes
  in genes that prevent uncontrolled cell growth
  (cancer growth stimulated by the absence of
  suppression)