Bacterial Transformation with (pGLO Plasmid)
Lab #8: Molecular Biology
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– Agriculture (pests, frost, & drought)– Bacteria (oil spills)– Gene therapy (sick cells into healthy cells)– Medicine (produce insulin & hormones)
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)
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
Chapters 18 & 19
Bacteria
Viruses & Operon Systems
Key Topics and Text Pgs to ReviewTopic Pgs.
Bacteria: Genetic recombination 346-350Plasmids & ConjugationTransformation (Lab #8)
Transposons: 351-352Lac Operon System 353-356Regulating Gene ExpressionViruses: DNA, RNA (retroviruses) 338-342Lytic & Lysogenic Cycle 337-339
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
Expected ResultsPLATES OBSERVATIONS
+pGlo
LB/amp
Many colonies with white appearance
Transformation observed (resistance to amp)
NO fluorescence (No arabinose present)
+pGlo
LB/amp/ara
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)
Introductory Questions #1) Briefly explain the differences between
Transformation, Conjugation, and Transduction. How are these three processes the same? (pgs. 348-349)
2) How is an “F plasmid” different from an “R plasmid”?
3) What are transposable elements and what do they do?
Introductory Questions #
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) Name three example of a virus that has DNA as its genetic material and three examples of Viruses with RNA as its genetic material.
5) Briefly explain what a vaccine is and what it does.
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. 352Specialized 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
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
Inactive Repressor-Lactose Present
Lac Operon Summary
• Beta-Galactosidase can then be made
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)
Inactive Repressor-Tryptophan Absent
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
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
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
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
Lytic & Lysogenic Cycles of a Virus(Lysogenic:host is not destroyed)
5 Classes of Viruses-Pg. 340
Examples of Common Viruses
DNA RNAHerpesvirus Ebola
Poxvirus Infuenza
Papovirus (warts) HIV
Measels, Mumps
Rabies
West Nile
HIV Infection (pgs 340-342)
HIV infection on a White Blood Cell
Lac Operon Summary
• Beta-Galactosidase can then be made
Key Concepts for Chapter 19
• Oncogenes & Proto-Oncogenes 370-373
• Tumor Supressor Genes
• McClintok’s transposons 375-376
Introductory Questions #
1) Why are transposons called “jumping genes”? What purpose do the insertion sequences play?
2) What is the difference between an oncogene and a tumor repressor gene?
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)