pglo tm bacterial transformation aequorea victoria two views of the hydromedusa aequorea victoria...
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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
Picture, Copyright © 2002 Pearson Education, Inc., publishiPicture, Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummingsng as Benjamin Cummings
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