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pGLO™ Transformation and Purification of

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Presentation on theme: "pGLO™ Transformation and Purification of"— Presentation transcript:

1

2 pGLO™ Transformation and Purification of
Green Fluorescent Protein (GFP)

3 Instructors Stan Hitomi Coordinator – Math & Science
Principal – Alamo School San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Bio-Rad Curriculum and Training Specialists: Sherri Andrews, Ph.D. Essy Levy, M.Sc. Leigh Brown, M.A. Instructors

4 Why Teach Bacterial Transformation and Protein Purification?
Powerful teaching tool Laboratory extensions Real-world connections Link to careers and industry Standards based

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6 pGLO™ Bacterial Transformation Kit
Bio-Rad pGLO Kit Advantages Standards-based Comprehensive curricula for inquiry-based investigations Compatible with 50 minute class periods Serves entire class of 32 students (up to 4 students per group) Cost-effective Success in student’s hands Safe Striking results!

7 Green Fluorescent Protein (GFP) Chromatography Kit
GFP Purification Kit Advantages Cloning in action Links to biomanufacturing Biopharmaceutical development Amazing visual results

8 Workshop Time Line Introduction Transform bacteria with pGLO plasmid
Purify GFP using column chromatography

9 Central Framework of Molecular Biology
DNA RNA Protein Trait

10 Links to Real-world GFP is a visual marker
Study of biological processes (example: synthesis of proteins) Localization and regulation of gene expression Cell movement Cell fate during development Formation of different organs Screenable marker to identify transgenic organisms

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12 Using GFP as a biological tracer
With permission from Marc Zimmer

13 pGLO Bacterial Transformation Kit

14 Transformation Procedure Overview
Day 1 10 Day 2

15 What is Transformation?
GFP Uptake of foreign DNA, often a circular plasmid Beta-lactamase Ampicillin Resistance

16 What is a plasmid? A circular piece of autonomously replicating DNA
Originally evolved by bacteria May express antibiotic resistance gene or be modified to express proteins of interest

17 Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

18 The Many Faces of Plasmids
Graphic representation Scanning electron micrograph of supercoiled plasmid

19 Gene Expression Beta Lactamase Ampicillin resistance
Green Fluorescent Protein (GFP) Aequorea victoria jellyfish gene araC regulator protein Regulates GFP transcription

20 Bacterial Transformation
Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids

21 Transcriptional Regulation
Lactose operon Arabinose operon pGLO plasmid

22 Transcriptional Regulation
RNA Polymerase Z Y A LacI Effector (Lactose) lac Operon B A D araC RNA Polymerase Effector (Arabinose) ara Operon

23 Gene Regulation B A D ara Operon ara GFP Operon GFP Gene
araC RNA Polymerase Effector (Arabinose) ara Operon RNA Polymerase araC ara GFP Operon GFP Gene Effector (Arabinose)

24 Methods of Transformation
Electroporation Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat-Shock Chemically-competent cells uptake DNA after heat shock

25 Transformation Procedure
Suspend bacterial colonies in Transformation solution Add pGLO plasmid DNA Place tubes on ice Heat-shock at 42°C and place on ice Incubate with nutrient broth Streak plates

26 Reasons for Performing Each Transformation Step?
Ca++ O Ca++ O P O Base O O CH2 Sugar Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates O Ca++ O P O Base O O CH2 Sugar OH

27 Why Perform Each Transformation Step?
Cell wall GFP 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression Beta-lactamase (ampicillin resistance)

28 What is Nutrient Broth? Luria-Bertani (LB) broth
Medium that contains nutrients for bacterial growth and gene expression Carbohydrates Amino acids Nucleotides Salts Vitamins

29 Grow? Glow? Follow protocol On which plates will colonies grow?
LB/Amp Follow protocol On which plates will colonies grow? Which colonies will glow? LB/Amp/Ara LB

30 Laboratory Quick Guide

31 GFP Electrophoresis Extension
LB/amp LB/amp/ara SDS PAGE sample preps are made from white and green colonies Bacterial lysates are prepared in Laemmli buffer Samples are loaded onto polyacrylamide gels

32 GFP Visualization-During & Post Electrophoresis
M W G M W G M W G Samples are electrophoresed Fluorescent GFP can be visualized during electrophoresis Coomassie stained gels allow for visualization of induced GFP proteins Fluorescent isoform Non-fluorescent isoform During Electrophoresis Post Electrophoresis Prestained bands + UV activated GFP Fluorescent bands Coomassie stained bands

33 Volume Measurement

34 GFP Chromatography Kit

35 GFP Purification Procedures Overview
Day 1 Day 2 Day 3

36 Why Use Chromatography?
To purify a single recombinant protein of interest from over 4,000 naturally occurring E. coli gene products.

37 Column Chromatography
Chromatography used for protein purification Size exclusion Ion exchange Hydrophobic interaction

38 Hydrophobic Interaction Chromatography: (HIC) Steps 1–3
Add bacterial lysate to column matrix in high salt buffer 2. Wash less hydrophobic proteins from column in low salt buffer Elute GFP from column with no salt buffer

39 Step 1: Hydrophobic Interaction Chromatography
bead Add bacterial lysate to column matrix in high salt buffer Hydrophobic proteins interact with column Salt ions interact with the less hydrophobic proteins and H2O N H + - O S N H + - O S

40 Step 2: Hydrophobic Interaction Chromatography
bead Wash less hydrophobic from column with low salt buffer Less hydrophobic E. coli proteins fall from column GFP remains bound to the column - + - + N H + - O S - +

41 Step 3: Hydrophobic Interaction Chromatography
bead Elute GFP from column by adding a no-salt buffer GFP Released from column matrix Flows through the column - + - + - + - + - +

42 Laboratory Quick Guide

43 Helpful Hints: Hydrophobic Interaction Chromatography
Add a small piece of paper to collection tube where column seats to insure column flow Rest pipet tip on side of column to avoid column bed disturbance when adding solutions Drain until the meniscus is just above the matrix for best separation

44 Webinars Enzyme Kinetics — A Biofuels Case Study
Real-Time PCR — What You Need To Know and Why You Should Teach It! Proteins — Where DNA Takes on Form and Function From plants to sequence: a six week college biology lab course From singleplex to multiplex: making the most out of your realtime experiments explorer.bio-rad.comSupportWebinars


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