Transcript Lac Operon - cloudfront.net

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
Applications of Genetic Transformation
• Used in many areas of Biotechnology
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Agriculture (pests, frost, & drought)
Bacteria (oil spills)
Gene therapy (sick cells into healthy cells)
Medicine (produce insulin & hormones)
Key Terms to Know
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DNA:
Bacteria:
Growth media:
Ampicillin:
Arabinose:
Heat shock
• GFP:
Plasmid
E. coli (strain: HB101K-12)
LB Broth (Luria & Bertani)
Antibiotic kills bacteria “amp”
Sugar source for energy & carbon
Process that increases permeability
of the cell membrane to DNA
Green Fluorescent Protein (w/UV)
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
Introductory Questions #4
1) After making the observations from our
transformations lab which plate(s) should not have any
bacteria growth? Which plate should contain the
glowing bacteria under UV light?
2) Briefly explain the differences between
Transformation, Conjugation, and Transduction. How
are these three processes the same? (pgs. 348-349)
3) How is an “F plasmid” different from an “R plasmid”?
4) What are transposable elements and what do they do?
See pg. 351-352.
5) What do insertion sequences code for? What type of
organisms are these sequences found?
6) How is are transposons different from insertion
sequences?
Bacterial Transformation with
(pGLO Plasmid)
Lab #9: Molecular Biology
Expected Results
PLATES
+pGlo
LB/amp
+pGlo
LB/amp/ara
OBSERVATIONS
Many colonies with white appearance
Transformation observed (resistance to amp)
NO fluorescence (No arabinose present)
Many transformed white colonies
Fluoresce bright green under UV light
-pGlo
LB/amp
(CONTROL)
No Bacterial growth present on the plate
No transformation
-pGlo
LB only
(CONTROL
Bacteria present with whitish colonies
(regeneration of the starter plate)
Chapters 18 & 19
Bacteria
Viruses & Operon Systems
Relative size Differences between of Viruses,
Prokaryotes, and Eukaryotes
Bacterial Reproduction of DNA
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.
Conjugation and the transfer of the F
Plasmid
Transduction
Detecting Genetic Recombination in
Bacteria
Insertion Sequences & Transposable Elements
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter20/animations.html#
• 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 and
Eukaryotes
See pg. 352
Specialized plasmids are constructed using these
sequences.
Jacob & Monod
• Discovered Lac Operon
– Nobel Prize for
Discovering Control of
Gene Expression
Regulation of a Metabolic Pathway
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
OPERON THEORY
• Operon = group of structural genes regulated as a
unit
• Several genes controlled by an operator site
Operon Complex
• RNA Polymerase must bind to the promoter site
and continue past the operator site to transcribe
mRNA
Key Topics for Ch. 18
Topic
Bacteria:
Genetic recombination
Plasmids & Conjugation
Transformation (Lab #9)
Transposons:
Lac Operon System
Regulating Gene Expression
Viruses: DNA, RNA (retroviruses)
Lytic & Lysogenic Cycle
Pgs.
346-350
351-352
353-356
334-342
337-339
Key Concepts for Chapter 19
• Review of DNA & Genome
• Oncogenes & Proto-Oncogenes
• Tumor Supressor Genes
359-362
370-373
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter20/animations.html#
• McClintok’s transposons
375-376
Introductory Questions #5
1) Name the two scientists that discovered the Lac
operon system.
2) How are repressible operons different from inducible
operons? Give an example of each.
3) What is the difference between an operator and a
promoter?
4) Why are transposons called “jumping genes”? What
purpose do the insertion sequences play?
5) Name three example of a virus that has DNA as its
genetic material and three examples of Viruses with
RNA as its genetic material.
6) Briefly explain what a vaccine is and what it does.
Repressible Operons (trp operon)
• 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)
Trp Operon when Tryptophan is Absent
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html#
INDUCIBLE Operons (ex. lac operon)
• 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
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
Inactive Repressor-Lactose Present
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html
Lac Operon
• In absence of glucose, cAMP levels are
HIGH, binding to CAP can occur
• Beta-Galactosidase is made
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
Lac Operon
Summary
DNA & RNA Viruses
Introductory Questions #5
1) Name the two scientists that discovered the Lac
operon system.
2) How are repressible operons different from inducible
operons? Give an example of each.
3) What is the difference between an operator and a
promoter?
4) Why are transposons called “jumping genes”? What
purpose do the insertion sequences play?
5) Name three example of a virus that has DNA as its
genetic material and three examples of Viruses with
RNA as its genetic material.
6) Briefly explain what a vaccine is and what it does.
Lytic & Lysogenic Cycles of a Virus
(Lysogenic:host is not destroyed)
5 Classes of Viruses-Pg. 340
Examples of Common Viruses
DNA
Herpesvirus
Poxvirus
Papovirus (warts)
RNA
Ebola
Infuenza
HIV
Measels, Mumps
Rabies
West Nile
HIV Infection (pgs 340-342)
HIV infection on a White Blood Cell
Invasion of a Virus
Vaccines-Pg. 343
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Harmless variants of the pathogen
Stimulate the immune system-produce antibodies
Smallpox has thought to be erradicated (polio)
Rubella, mumps, hepatitis B, other viral diseases
The Biology of Cancer
Oncogenes & Proto-oncogenes
Molecular Biology of Cancer
pgs. 370-371
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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)
Video #3-Cell Biology & Cancer
• Write 10 Statements and three Questions
from the video.
• Be sure to comment on what the differences
are between Oncogenes and Tumor
supressor genes.