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)