pGLO Lab Background Information
-
Upload
klipp182174 -
Category
Documents
-
view
2.700 -
download
3
Transcript of pGLO Lab Background Information
![Page 1: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/1.jpg)
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.
![Page 2: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/2.jpg)
Aequorea victoria: Source of “glowing gene” for this experiment
![Page 3: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/3.jpg)
Jellyfish Gene put into Other CrittersJellyfish Gene put into Other Critters
![Page 4: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/4.jpg)
OutlineOutline
• Overview • Bacteria and Plasmids• Transformation• The pGLO Plasmid• Experimental Procedures• Extension Activities
![Page 5: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/5.jpg)
OverviewOverview
![Page 6: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/6.jpg)
What is Bacterial What is Bacterial Transformation?Transformation?
Taking up of DNA from the environment by bacterial cells
![Page 7: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/7.jpg)
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
![Page 8: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/8.jpg)
Bacteria and PlasmidsBacteria and Plasmids
![Page 9: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/9.jpg)
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
![Page 10: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/10.jpg)
Bacterial Cells and DNABacterial Cells and DNA
Chromosomal DNA
Chromosomal
Bacterial cell
Plasmid DNA
![Page 11: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/11.jpg)
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
![Page 12: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/12.jpg)
TransformationTransformation
![Page 13: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/13.jpg)
Bacterial Transformation
Plasmids
Chromosomal DNA
Bacterial Cell
The uptake of DNA
![Page 14: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/14.jpg)
Methods of transformation
ElectroporationElectrical shock makes cell membranes permeable to DNA
Calcium Chloride/Heat ShockChemically-competent cells uptake DNA after heat shock
![Page 15: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/15.jpg)
The pGLO PlasmidThe pGLO Plasmid
![Page 16: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/16.jpg)
pGLOori
blaGFP
araC
pGLO Plasmid
bla genebeta-lactamase enzyme
Ampicillin resistance
GFP geneGreen Fluorescent ProteinAequorea victoria jellyfish
araC geneRegulates GFP transcription
oriAllows plasmid replication
![Page 17: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/17.jpg)
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
![Page 18: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/18.jpg)
Experimental ProceduresExperimental Procedures
![Page 19: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/19.jpg)
Transformation Procedures
+CaCl2 +CaCl2
![Page 20: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/20.jpg)
Transformation Procedures
![Page 21: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/21.jpg)
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++
![Page 22: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/22.jpg)
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
![Page 23: pGLO Lab Background Information](https://reader030.fdocuments.us/reader030/viewer/2022020217/54674cd3af795974338b55d8/html5/thumbnails/23.jpg)
BiotechnologyBiotechnologyExplorer ProgramExplorer Program
Serious About Science Education