Module based on a kit from Bio-Rad Laboratories, Inc. · Module based on a kit from Bio-Rad...
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This Little Light of Mine: This Little Light of Mine: Transform bacteria with a Jellyfish gene to make them glow Module based on a kit from Bio-Rad Laboratories, Inc. Adapted by Dan Murray from a presentation by Stan Hitomi Monte Vista High School, Danville, CA. Kirk Brown Tracy High School, Tracy, CA.
Transcript of Module based on a kit from Bio-Rad Laboratories, Inc. · Module based on a kit from Bio-Rad...
This Little Light of Mine:This Little Light of Mine:Transform bacteria with a Jellyfish
gene to make them glow
Module based on a kit from Bio-Rad Laboratories, Inc.
Adapted by Dan Murray from a presentation by
Stan HitomiMonte Vista High School, Danville, CA.
Kirk BrownTracy High School, Tracy, CA.
Aequorea victoria: Source of “glowing gene” for this experiment
Jellyfish Gene put into Other CrittersJellyfish Gene put into Other Critters
OutlineOutline
• Overview • Bacteria and Plasmids• Transformation• The pGLO Plasmid• Experimental Procedures• Extension Activities
OverviewOverview
What is Bacterial What is Bacterial Transformation?Transformation?
Taking up of DNA from the environment by bacterial cells
Bacterial Transformation LabBacterial Transformation Lab
• Only cells which obtained plasmid DNA will grow… and glow
• Cell/DNA mix is plated on nutrient agar with antibiotic
• Cells take up plasmid
• Bacterial Cells and plasmid DNA are mixed
Bacteria and PlasmidsBacteria and Plasmids
What is a plasmid?What is a plasmid?
Small circular DNA molecule Replicates autonomously Originally evolved in bacteriaMay contain antibiotic
resistance gene or be modified to contain other genes
bla is an ampicillin resistance gene
ori
bla
Bacterial Cells and DNABacterial Cells and DNA
Chromosomal DNA
Chromosomal
Bacterial cell
Plasmid DNA
Growth of Bacteria Growth of Bacteria on Plateson Plates
Agarose in Petri dish = plate
bacteria
Incubate at 37°CIf few
cells growIf many
cells grow
colonies lawn
TransformationTransformation
Bacterial Transformation
Plasmids
Chromosomal DNA
Bacterial Cell
The uptake of DNA
Methods of transformation
ElectroporationElectrical shock makes cell membranes permeable to DNA
Calcium Chloride/Heat ShockChemically-competent cells uptake DNA after heat shock
The pGLO PlasmidThe pGLO Plasmid
pGLOori
blaGFP
araC
pGLO Plasmid
bla genebeta-lactamase enzyme
Ampicillin resistance
GFP geneGreen Fluorescent ProteinAequorea victoria jellyfish
araC geneRegulates GFP transcription
oriAllows plasmid replication
pGLO
blaGFP
pGLO Plasmid: Most Important Components
bla geneBacteria with this gene grow in the presence of ampicillin
GFP geneBacteria with this gene glow under near UV light
Experimental ProceduresExperimental Procedures
Transformation Procedures
+CaCl2 +CaCl2
Transformation Procedures
Reasons for Each Transformation Step
CaCl2 treatment
Positive charge of Ca2+
ions neutralizes: • negative charge of DNA
phosphates • negative charge of
membrane phospholipids
Ca++
Ca++
OCH2
O
P O
OO Base
CH2
O
PO
O
O
Base
OH
Sugar
Sugar
OCa++
Incubation on ice slows fluid cell membranes
Heat-shock increases permeability of cell membrane
Nutrient broth incubationallows beta lactamase expression
Reasons for Each Transformation Step
Transformation Results
Only cells getting pGLO plasmid grow and glow
All cells grow since there is no antibiotic on the plate
Without pGLO plasmid, nothing can grow
All cells grow since there is no antibiotic on the plate
Extension ActivitiesExtension Activities
Extension Activity I: Transcriptional Regulation
Arabinose controls expression of GFP gene:
Glowing Bacteria from Transformation
Plate with Arabinose
Plate without Arabinose
Transfer Bacteria
Incubate overnight @ 37°C
Extension Activity I: Transcriptional Regulation
−arabinose = no glow+arabinose = glow
Plate with Arabinose
Plate without Arabinose
After overnight incubation
Transcriptional Regulation of GFP by Arabinose
araC GFP Gene
araC GFP Gene
RNA Polymerase
Arabinose
araC
GFP Gene
araC repressor blocks transcription
Arabinose binds repressor, changing its conformation
Altered repressor leaves DNA, RNA polymerase can perform transcription
Extension Activity II: Tweaking the Transformation Protocol
Test effect of various components of the transformation protocol:
plate ampicillin concentration plate arabinose concentration amount of plasmid DNA used in the experiment amount of cells used in the experiment length of time cells/DNA mix is kept at 42°C during the experiment
Compare results with number of colonies obtained during the normal protocol
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