Download presentation
Published byChristopher Spinney Modified over 10 years ago
1
BioRad pGLO: Transform bacteria with a Jellyfish gene to make them glow
Module based on a kit from Bio-Rad Laboratories, Inc. Thank you to : Adapted by Dan Murray from a presentation by Stan Hitomi Monte Vista High School, Danville, CA. Kirk Brown Tracy High School, Tracy, CA.
2
Source of “glowing gene” for this experiment
Aequorea victoria: Source of “glowing gene” for this experiment
3
Jellyfish Gene put into Other Critters
4
Outline Overview Bacteria and Plasmids Transformation The pGLO Plasmid
Experimental Procedures Extension Activities
5
Overview
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 mixed Cells take up plasmid Cell/DNA mix is plated on nutrient agar with antibiotic Only cells which obtained plasmid DNA will grow… and glow
8
Bacteria and Plasmids
9
What is a plasmid? Small circular DNA molecule Replicates autonomously
Originally evolved in bacteria May contain antibiotic resistance gene or be modified to contain other genes bla is an ampicillin resistance gene ori bla
10
Bacterial Cells and DNA
Chromosomal Bacterial cell Chromosomal DNA Plasmid DNA
11
Growth of Bacteria on Plates
Agarose in Petri dish = plate Incubate at 37C If few cells grow If many cells grow colonies lawn
12
Transformation
13
Bacterial Transformation
The uptake of DNA Bacterial Cell Chromosomal DNA Plasmids
14
Methods of transformation
Electroporation Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat Shock Chemically-competent cells uptake DNA after heat shock
15
The pGLO Plasmid
16
pGLO Plasmid bla gene GFP gene araC gene ori beta-lactamase enzyme
Ampicillin resistance GFP gene Green Fluorescent Protein Aequorea victoria jellyfish araC gene On/off switch that reacts to arabinose ori Allows plasmid replication pGLO ori bla GFP araC
17
pGLO Plasmid: Most Important Components
bla gene Bacteria with this gene grow in the presence of ampicillin GFP gene Bacteria with this gene glow under near UV light pGLO GFP bla
18
Experimental Procedures
19
Transformation Procedures
+CaCl2 +CaCl2
20
Transformation Procedures
21
Reasons for Each Transformation Step
Ca++ O CH2 P Base OH Sugar CaCl2 treatment Positive charge of Ca2+ ions neutralizes: negative charge of DNA phosphates negative charge of membrane phospholipids
22
Reasons for Each Transformation Step
Incubation on ice slows fluid cell membranes Heat-shock increases permeability of cell membrane Nutrient broth incubation allows beta lactamase expression video
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 glow CONTROL 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
26
Extension Activities
27
Extension Activity I: Transcriptional Regulation
Arabinose controls expression of GFP gene: Transfer Bacteria Glowing Bacteria from Transformation Plate with Arabinose Plate without Arabinose Incubate 37C
28
Extension Activity I: Transcriptional Regulation
arabinose = no glow +arabinose = glow After overnight incubation Plate with Arabinose Plate without Arabinose
29
Transcriptional Regulation of GFP by Arabinose
araC GFP Gene araC repressor blocks transcription Arabinose araC GFP Gene Arabinose binds repressor, changing its conformation RNA Polymerase araC GFP Gene Altered 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 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 42C during the experiment Compare results with number of colonies obtained during the normal protocol
31
Biotechnology Explorer Program Serious About Science Education
Similar presentations
© 2025 SlidePlayer.com. Inc.
All rights reserved.