pglo tm bacterial transformation aequorea victoria two views of the hydromedusa aequorea victoria...

pGLOpGLOTMTM Bacterial Transformation Bacterial Transformation

Aequorea victoria Two views of the hydromedusa Aequorea victoria from Friday Harbor, Washington, copyright Claudia .E. Mills 1999

pGlo ConceptspGlo Concepts

Genetic TransformationGenetic Transformation

Gene RegulationGene Regulation

Genetic SelectionGenetic Selection

DNA RNA Protein Trait

The Central Dogma of The Central Dogma of Molecular BiologyMolecular Biology

AdvantagesAdvantages The DNA can The DNA can

retain integrityretain integrity The RNA step The RNA step

allows allows amplificationamplification

Multiple steps Multiple steps allow multiple allow multiple points of points of controlcontrol

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What is transformation?What is transformation?

Uptake of foreign DNAUptake of foreign DNA, often a , often a circular plasmidcircular plasmid

Plasmid

Bacterial chromosomal DNA

Cell wall

What is a plasmid?What is a plasmid?

The most simple bacterial The most simple bacterial vectorvector: a DNA molecule used to : a DNA molecule used to insert foreign DNA into a host insert foreign DNA into a host cell cell

A A circularcircular piece of piece of autonomously replicatingautonomously replicating DNA DNA

Plasmids are like mini-Plasmids are like mini-chromosomeschromosomes

Originally evolved by bacteriaOriginally evolved by bacteria May express antibiotic May express antibiotic

resistance gene or be modified resistance gene or be modified to express proteins of interestto express proteins of interest

ori

bla

PlasmidsPlasmids Differ from chromosomesDiffer from chromosomes

Range in size from 1,000—200,000 bp (base Range in size from 1,000—200,000 bp (base pairs) pairs)

One or more copies per cell, “stringent” vs. One or more copies per cell, “stringent” vs. “relaxed”“relaxed” : <12 is normal, but can range from ~5 : <12 is normal, but can range from ~5 to 700 copies per cellto 700 copies per cell

Not all bacteria have plasmidsNot all bacteria have plasmids FunctionsFunctions

Cryptic – no known functionCryptic – no known function Phenotypic – antibiotic resistance, conjugative, Phenotypic – antibiotic resistance, conjugative,

virulence, etc.virulence, etc.

Inserting DNA into a PlasmidInserting DNA into a Plasmid

The pGlo plasmidThe pGlo plasmid

Beta LactamaseBeta Lactamase Ampicillin resistanceAmpicillin resistance

araC regulator proteinaraC regulator protein Regulates GFP Regulates GFP

transcriptiontranscription

Green Fluorescent Green Fluorescent ProteinProtein Aequorea victoriaAequorea victoria

jellyfish genejellyfish gene

pGLOori

blaGFP

araC

Ampicillin Action and ResistanceAmpicillin Action and Resistance

Antibiotics have various methods of interfering with Antibiotics have various methods of interfering with bacterial growth: inhibiting cell wall biosynthesis or bacterial growth: inhibiting cell wall biosynthesis or blocking protein synthesisblocking protein synthesis

Ampicillin inhibits Ampicillin inhibits peptidoglycan synthesispeptidoglycan synthesis: the polymer : the polymer consisting of sugars and amino acids that forms a consisting of sugars and amino acids that forms a homogeneous layer outside the plasma membrane of homogeneous layer outside the plasma membrane of eubacteria. eubacteria.

The pAMP resistance protein, The pAMP resistance protein, ββ-lactamase-lactamase, cleaves the , cleaves the ββ-lactam ring of ampicillin molecules, which leaves -lactam ring of ampicillin molecules, which leaves them unable to interfere with them unable to interfere with peptidoglycan synthesispeptidoglycan synthesis

Antibiotic resistance genes also have different Antibiotic resistance genes also have different mechanisms: chemically modifying target antibiotics or mechanisms: chemically modifying target antibiotics or preventing transport of antibiotics through the cell preventing transport of antibiotics through the cell membranemembrane

Gene Regulation: Bacterial OperonsGene Regulation: Bacterial Operons

The Lactose (Lac) OperonThe Lactose (Lac) Operon

Arabinose OperonArabinose Operon

RNA Polymerase

A more detailed description of the Arabinose Operon:http://www.mun.ca/biochem/courses/3107/Topics/Ara_operon.html

B A DaraC

Effector (Arabinose)

araC B A D

Promotor (PBAD)

DNA binding Protein: Represses Transcription

Genes coding for digestive enzymes

B A DaraC

Transcription

Ara-GFP OperonAra-GFP Operon

RNA Polymerase

araC GFP Gene

araC GFP Gene

araC GFP Gene

Effector (Arabinose)

Promotor (PBAD)

Transcription

GFP only produced in the GFP only produced in the presence of Arabinosepresence of Arabinose

Genes coding for Genes coding for digestive enzymes have digestive enzymes have been replaced by the GFP been replaced by the GFP gene: no metabolism of gene: no metabolism of arabinosearabinose

How Does it How Does it GLOWGLOW??

Unique 3-D Unique 3-D Structure of GFPStructure of GFP

Resonates when Resonates when exposed to exposed to ultraviolet lightultraviolet light

Gives off energy in Gives off energy in the form of green the form of green fluorescent lightfluorescent light

Learn more about GFP:Learn more about GFP:http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm

Picture, Copyright © 2002 Pearson Education, Inc., publishiPicture, Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummingsng as Benjamin Cummings

How does it work?How does it work?

GFP

Beta lactamase(ampicillin resistance)

pGlo Plasmids

Bacterial chromosomal DNA

Cell wall

Transform bacteria Transform bacteria with the pGlo with the pGlo plasmid and grow plasmid and grow under various under various conditionsconditions

Methods of Methods of transformationtransformation

DNA molecules are too large to easily DNA molecules are too large to easily diffuse or be transported though the diffuse or be transported though the cell membranecell membrane

ElectroporationElectroporation Electrical shock makes cell Electrical shock makes cell

membranes permeable to DNAmembranes permeable to DNA

Calcium Chloride/Heat ShockCalcium Chloride/Heat Shock Chemically-competent cells uptake Chemically-competent cells uptake

DNA after heat shockDNA after heat shock

ElectroporationElectroporation

TransformationTransformation

Transformation Procedure: Transformation Procedure: OverviewOverview

Suspend bacterial colonies in Suspend bacterial colonies in

Transformation SolutionTransformation Solution

Add Add pGLO plasmid DNA pGLO plasmid DNA

Place tubes on Place tubes on iceice

Heat shockHeat shock at 42 at 42ooC and place on C and place on iceice

Incubate withIncubate with LB LB nutrient brothnutrient broth

Streak Streak platesplates

Why perform each step?Why perform each step?

CaClCaCl22 treatment on ice treatment on ice crytallizes fluid membranes and crytallizes fluid membranes and stabilizes distribution of charged stabilizes distribution of charged moleculesmolecules

CaClCaCl22 Transformation Transformation solution provides Casolution provides Ca++ ++

cations cations thatthat neutralize the neutralize the repulsive negative charges of the repulsive negative charges of the phosphate backbone of the DNA phosphate backbone of the DNA and the phospholipids of the cell and the phospholipids of the cell membrane, allowing the DNA to membrane, allowing the DNA to enter the cellsenter the cells

Ca++

Ca++

OCH2

O

P O

O

O Base

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

OCa++

Picture, Copyright © 2002 Pearson Education, Inc., publishiPicture, Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummingsng as Benjamin Cummings

Why perform each step?Why perform each step?

Heat-shockHeat-shock increases permeability increases permeability of cell membraneof cell membrane

Luria-Bertani Luria-Bertani Nutrient broth Nutrient broth incubation incubation allows allows beta lactamase beta lactamase expressionexpression

Beta lactamase(ampicillin resistance)pGlo

Plasmids

Bacterial chromosomal DNA

Cell wall

Gene selectionGene selection

Grow transformed bacteria and control Grow transformed bacteria and control bacteria under various conditions.bacteria under various conditions.

On which plates will colonies On which plates will colonies grow?grow? Which colonies will Which colonies will glowglow??

The pGlo The pGlo SystemSystem

Timing is important…be efficient!!

Mix contents before pipetting!!!

A film of plasmid must be on the loop!

Sterile TechniqueSterile Technique

Bacteria are Bacteria are UBIQUITOUS…they are UBIQUITOUS…they are found EVERYWHERE!found EVERYWHERE!

Sterile technique refers Sterile technique refers to procedures that to procedures that reduce the possibility of reduce the possibility of contamination…these contamination…these techniques protect YOU, techniques protect YOU, your CULTURES and your CULTURES and REAGENTS, and LAB REAGENTS, and LAB EQUIPMENTEQUIPMENT

pGlo Lab ConsiderationspGlo Lab Considerations

Teacher ConsiderationsTeacher Considerations

Work surfacesWork surfaces HandsHands GlasswareGlassware AgarAgar Petri platesPetri plates Inoculation loopsInoculation loops Solutions/CulturesSolutions/Cultures PipettesPipettes

Student ConsiderationsStudent Considerations

Work surfacesWork surfaces HandsHands E.coli starter platesE.coli starter plates Assorted agar platesAssorted agar plates Inoculation loopsInoculation loops Solutions/Test tubesSolutions/Test tubes PipettesPipettes

Closing ConsiderationsClosing Considerations

ALWAYS decontaminate you work ALWAYS decontaminate you work surfaces with a disinfecting solution: surfaces with a disinfecting solution: 20ml of liquid household bleach in 1L of 20ml of liquid household bleach in 1L of tap water.tap water. Spray the solution on work surfaces and let Spray the solution on work surfaces and let

stand for 2 minutes and wipe awaystand for 2 minutes and wipe away

ALWAYS thoroughly scrub hands for at ALWAYS thoroughly scrub hands for at least 20 seconds with soap and hot water least 20 seconds with soap and hot water before leaving the lab areabefore leaving the lab area

Volume MeasurementVolume Measurement

Extension ActivitiesExtension Activities

Calculate Transformation EfficiencyCalculate Transformation Efficiency This protocol has been determined to have This protocol has been determined to have

a transformation efficiency between 8.0 x a transformation efficiency between 8.0 x 10102 2 and 7.0 x 10and 7.0 x 1033 (128-1120 transformed (128-1120 transformed colonies)colonies)

Students explore reasons for various resultsStudents explore reasons for various results