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: Bacteria: Growth media: Ampicillin: Arabinose: Heat shock 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)

GFP:

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 Review

Topic Bacteria: Genetic recombination Plasmids & Conjugation Transformation (Lab #8) Transposons: Lac Operon System Regulating Gene Expression Viruses: DNA, RNA (retroviruses) Lytic & Lysogenic Cycle Pgs. 346-350

351-352 353-356 338-342 337-339

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 Results
PLATES
+pGlo LB/amp +pGlo LB/amp/ara -pGlo LB/amp (CONTROL) -pGlo LB only (CONTROL

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

No Bacterial growth present on the plate No transformation 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. 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

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

BetaGalactosidase 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 betagalactosidase
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
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

Lac Operon Summary

BetaGalactosidase can then be made

Key Concepts for Chapter 19

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

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)