Presentation on theme: "Module based on a kit from Bio-Rad Laboratories, Inc. Thank you to :"— Presentation transcript:
1 BioRad pGLO: Transform bacteria with a Jellyfish gene to make them glow Module based on a kit fromBio-Rad Laboratories, Inc.Thank you to :Adapted by Dan Murray from a presentation byStan HitomiMonte Vista High School, Danville, CA.Kirk BrownTracy High School, Tracy, CA.
2 Source of “glowing gene” for this experiment Aequorea victoria:Source of “glowing gene” for this experiment
6 What is Bacterial Transformation? Taking up of DNA from the environment by bacterial cells
7 Bacterial Transformation Lab Bacterial Cells and plasmid DNA are mixedCells take up plasmidCell/DNA mix is plated on nutrient agar with antibioticOnly cells which obtained plasmid DNA will grow… and glow
9 What is a plasmid? Small circular DNA molecule Replicates autonomously Originally evolved in bacteriaMay contain antibiotic resistance gene or be modified to contain other genesbla is an ampicillin resistance geneoribla
10 Bacterial Cells and DNA ChromosomalBacterial cellChromosomal DNAPlasmid DNA
11 Growth of Bacteria on Plates Agarose in Petri dish = plateIncubate at 37CIf fewcells growIf manycells growcolonieslawn
21 Reasons for Each Transformation Step Ca++OCH2PBaseOHSugarCaCl2 treatmentPositive charge of Ca2+ ions neutralizes:negative charge of DNA phosphatesnegative charge of membrane phospholipids
22 Reasons for Each Transformation Step Incubation on ice slows fluid cell membranesHeat-shock increases permeability of cell membraneNutrient broth incubation allows beta lactamase expressionvideo
23 Purpose of each plate -pGLO/LB = Control -pGLO/LB/amp = tests the effect of ampicillin+pGLO/LB/amp = shows that ampicillin resistance has been acquired+pGLO/LB/amp/ara = shows that both traits have been acquired
24 Transformation Results Only cells getting pGLO plasmid grow and glowCONTROLAll cells grow since there is no antibiotic on the plateWithout pGLO plasmid, nothing can growAll cells grow since there is no antibiotic on the plate
29 Transcriptional Regulation of GFP by Arabinose araCGFP GenearaC repressor blocks transcriptionArabinosearaCGFP GeneArabinose binds repressor, changing its conformationRNA PolymerasearaCGFP GeneAltered repressor leaves DNA, RNA polymerase can perform transcription
30 Extension Activity II: Tweaking the Transformation Protocol Test effect of various components of the transformation protocol:plate ampicillin concentrationplate arabinose concentrationamount of plasmid DNA used in the experimentamount of cells used in the experimentlength of time cells/DNA mix is kept at 42C during the experimentCompare results with number of coloniesobtained during the normal protocol
31 BiotechnologyExplorer ProgramSerious About Science Education
Your consent to our cookies if you continue to use this website.