Bio-Rad Laboratories, Inc. · NYSE:BIO
Bio-Rad Laboratories — Investor Presentation (February 2024)
96 slides from Bio-Rad Laboratories's investor presentation, February 2024
Slide 1: pGLO Bacterial Transformation for General Biology
pGLO Bacterial Transformation for General Biology Background and Student Protocol BIO-RAD is a trademark of Bio-Rad Laboratories, Inc. All trademarks used herein are the property of their respective owner. © 2023 Bio-Rad Laboratories, Inc.
Slide 2: Slide Deck Overview
Slide Deck Overview Background Student Activity Results and Information or Lab Protocol Troubleshooti ng Tips & Notes
Slide 3: Background Information
Background Information Genetic engineering, GFP, plasmids, bacterial transformation, and selection
Slide 4: Setting the stage
Setting the stage
Slide 5: Insulin timeline
Insulin timeline First patient (14 years old) is treated with insulin Insulin from cow and pig Previously, diabetes was pancreas continues to be a death sentence – there used for decades was no treatment or 1921 1923 1978 cure 1922 1930 s- Frederick Banting Eli Lilly begins First genetically figures
Slide 6: Cost per gram
Cost per gram Gold Insulin Human Granulocy Growth te Colony Hormone Stimulatin g Factor $63 $8800 $580,000 $1 million+
Slide 7: Other Recombinant Drugs
Other Recombinant Drugs Molecule / Drug (Brand) Indications Cell Factory Mechanism Cytokine — stimulates Filgrastim Acute lymphocytic growth of white blood E. coli (Neupogen) leukemia cells AIDS-related Interferon alfa- Recombinant interferon Kaposi’s sarcoma, E. coli 2a - antiviral chronic hepatiti
Slide 8: How do we produce drugs like insulin?
How do we produce drugs like insulin? 1 Clone human insulin 2 Transform 3 Select transformed 4 Culture bacteria and gene into plasmid bacteria with bacteria induce gene expression plasmid Purify protein Analyze purification 5 Lyse cells 6 7 8 Assay protein activity with with SDS-PAGE chromatography
Slide 9: What did you notice?
What did you notice? www.bio-rad.com/glow • About the bacteria under the UV light? • About the difference between the +pGLO and –pGLO bacteria?
Slide 10: E. Coli colonies
E. Coli colonies The bacterial growth you see on the plate is made of millions of individual bacterial (E. coli) cells.
Slide 11: Aequorea victoria
Aequorea victoria • Similarities and differences between these glowing jellyfish and bacteria? • How do you think jellyfish make green fluorescent protein (GFP)? Visible light UV light
Slide 12: Green Fluorescent Protein (GFP)
Green Fluorescent Protein (GFP) GFP allows us to visualize protein expression with UV light
Slide 13: GFP as a visual tracer / marker
GFP as a visual tracer / marker • Study of biological processes (such as protein synthesis) • Localization and regulation of gene expression • Cell movement Sparse granule cells | i nfected with HSV, expr essing gfp | Jason Snyd • Cell fate during development er | Flickr • Formation of different org
Slide 14: GFP as a visual tracer
GFP as a visual tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer
Slide 15: GFP (and other variants) animal – GloFish!
GFP (and other variants) animal – GloFish!
Slide 16: Genetic engineering / transformation
Genetic engineering / transformation Research Agriculture Medicine transgenic bacteria to corn, Hawaiian papaya, synthetic insulin, human produce DNA and soybeans, cotton, sugar growth hormones, proteins beets, canola, alfalfa vaccines, CAR-T
Slide 17: Genetic engineering using plasmids
Genetic engineering using plasmids bacterium chromosome plasmids
Slide 18: Generic plasmid
Generic plasmid ori origin of replication pGene allows bacteria to ric make copies of plasmid gene gene of interest cloned into plasmid
Slide 19: Cloning a gene
Cloning a gene 1 Isolate gene of Cut plasmid with same 2 interest and cut with restriction enzymes restriction enzymes (or use PCR) 3 Insert gene into plasmid using DNA ligase
Slide 20: Bacterial transformation
Bacterial transformation Genetic transformation occurs when a cell takes up DNA and expresses the genes on that DNA. Many different types of cells can be transformed – plants, animals, human, bacteria.
Slide 21: Bacteria divide and new bacteria get plasmids
Bacteria divide and new bacteria get plasmids
Slide 22: Genes get transcribed and translated
Genes get transcribed and translated transcription translation protein
Slide 23: Activity: Model recombinant GFP bacteria
Activity: Model recombinant GFP bacteria
Slide 24: Plasmid with gene for GFP
Plasmid with gene for GFP ori origin of replication allows bacteria to make copies gfp gene of plasmid encodes Green Fluorescent Protein (GFP)
Slide 25: Selection of transformed bacteria
Selection of transformed bacteria
Slide 26: Selection of transformed bacteria
Selection of transformed bacteria
Slide 27: Plasmids can carry multiple genes
Plasmids can carry multiple genes
Slide 28: β-lactamase gene for antibiotic resistance
β-lactamase gene for antibiotic resistance ori origin of replication allows bacteria to make copies of plasmid ampr (or bla) gene gfp gene encodes Green encodes beta-lactamase, Fluorescent Protein an enzyme that breaks (GFP) down ampicillin (an antibiotic)
Slide 29: Transformation with plasmid with ampr
Transformation with plasmid with ampr
Slide 30: β-lactamase produced
β-lactamase produced
Slide 31: Bacteria plated on ampicillin plates
Bacteria plated on ampicillin plates
Slide 32: β-lactamase breaks down ampicillin
β-lactamase breaks down ampicillin
Slide 33: Bacteria that aren’t transformed don’t grow
Bacteria that aren’t transformed don’t grow
Slide 34: Transformed bacteria grow into colonies
Transformed bacteria grow into colonies
Slide 35: pGLO plasmid (simplified)
pGLO plasmid (simplified) ori origin of replication pGLO allows bacteria to make copies of plasmid ampr (or bla) gfp gene gene encodes Green encodes beta-lactamase, Fluorescent Protein an enzyme that breaks (GFP) down ampicillin (an antibiotic)
Slide 36: pGLO plasmid (simplified)
pGLO plasmid (simplified) pGLO gfp ampr
Slide 37: Student Lab Protocol – Day 1
Student Lab Protocol – Day 1 Bacterial transformation with pGLO plasmid
Slide 38: Lab protocol - transformation
Lab protocol - transformation 1. You have 2 tubes with 250 μl of transformation solution. Label the top of one tube + pGLO and the other – pGLO . Add your group ID or initials to each tube. 2. Put both tubes on ice.
Slide 39: Lab protocol - transformation
Lab protocol - transformation 3. Use a sterile loop to scrape 2-4 single E. coli colonies from the surface of the starter plate. Transfer the loop into the + pGLO tube and swirl it in the transformation solution to disperse the bacteria. Close the tube and place it back on ice. 4. Use a new loop and
Slide 40: Lab protocol - transformation
Lab protocol - transformation 5. Dip a loop into the pGLO plasmid vial, check for a film across the circle, and swirl the plasmid in the +pGLO tube. Do not add any plasmid into the – pGLO tube. 6. Incubate the tubes on ice for 10 minutes. Make sure the tubes are in full contact with the ice.
Slide 41: Lab protocol - transformation
Lab protocol - transformation 7. Label plates as shown here: • Draw a line down the center of each plate • Write + pGLO on one side of each plate, and – pGLO on the other. Note: Plates should be labeled on the bottom (agar side) around the edge, in small, neat writing, so the bacteria colonies can b
Slide 42: Look at the plasmid under UV light
Look at the plasmid under UV light What color is the DNA? Can you see any green?
Slide 43: Background Information
Background Information Bacterial transformation steps
Slide 44: DNA is negatively charged
DNA is negatively charged
Slide 45: CaCl2 transformation solution
CaCl2 transformation solution negative charges plasmid plasma membran e
Slide 46: CaCl2 transformation solution
CaCl2 transformation solution Ca2+ shields negative charges
Slide 47: Incubate on ice for 10 min
Incubate on ice for 10 min Ca2+ shields negative charges
Slide 48: Heat shock at 60°C for 50 sec
Heat shock at 60°C for 50 sec Pores form in membrane
Slide 49: Incubate on ice for 2 min
Incubate on ice for 2 min
Slide 50: LB broth
LB broth LB (lysogeny broth or Luria Bertani) broth is like chicken noodle soup for bacteria. It has all the nutrients bacteria need to grow: • Carbohydrates • Amino acids • Nucleotides • Salts • Vitamins
Slide 51: LB recovery
LB recovery During recovery, E. coli repair their cell walls and express the antibiotic resistance gene so they can grow on ampicillin plates.
Slide 52: Lab protocol - transformation
Lab protocol - transformation 8. Heat shock: get your timers ready! Transfer the tubes directly from the ice into a 60°C water bath or dry bath for exactly 50 sec, then immediately place them back on ice. 9. Incubate tubes on ice for 2 min. Note: Heat shock should be QUICK! Straight from ice, into h
Slide 53: Lab protocol - transformation
Lab protocol - transformation 10 Remove the tubes from ice . and pipet 250 μl of LB broth into each tube. Incubate at room temperature for at least 10 min. Note: Bacteria can be left at room temperature overnight before proceeding to the next step.
Slide 54: Activity: Predict transformation results
Activity: Predict transformation results Colonie Law s typically Bacteria All the nindividual grow in colonies that colonies have arise from 1 bacteria merged to form a dividing over and uniform layer of over. bacterial growth.
Slide 55: Activity: Predict transformation results
Activity: Predict transformation results
Slide 56: Lab protocol - transformation
Lab protocol - transformation 11 Flick tubes to mix. Pipet . one drop from each tube onto the appropriately half of each plate. Use a new pipet for each tube.
Slide 57: Lab protocol - transformation
Lab protocol - transformation 12 Use a new loop to spread . the bacteria on each half of each plate. Use a fresh loop each time. 13. Stack plates upside down (agar side up) and incubate overnight at 37°C or at room temperature for 2 days.
Slide 58: Results & Analysis After Day 1
Results & Analysis After Day 1
Slide 59: Results after day 1
Results after day 1
Slide 60: Results after day 1
Results after day 1 Do you think the gene for GFP was successfully transformed into any bacteria? Explain your reasoning.
Slide 61: Results after day 1 under regular light
Results after day 1 under regular light - + - + LB LB/amp
Slide 62: Results after day 1 under UV – no glow!
Results after day 1 under UV – no glow! - + - + LB LB/amp
Slide 63: Background Information
Background Information Control of gene expression with arabinose operon
Slide 64: pGLO plasmid – regulation of GFP expression
pGLO plasmid – regulation of GFP expression araC gene encodes the AraC repressor ori protein origin of replication pGLO pBAD promoter allows bacteria to landing site for RNA polymerase make copies of to transcribe downstream genes plasmid ampr (or bla) gene gfp gene encodes Green encodes beta-lactam
Slide 65: Without arabinose in the media
Without arabinose in the media OFF
Slide 66: With arabinose in the media
With arabinose in the media ON
Slide 67: Arabinose and AraC
Arabinose and AraC Arabinose (inducer) AraC acts as a repressor When arabinose binds AraC, the in the absence of AraC conformation arabinose changes, activating the expression of the araBAD genes that metabolize arabinose
Slide 68: Arabinose operon (araBAD operon)
Arabinose operon (araBAD operon) RNA polymerase OFF AraC Without arabinose, AraC acts as a repressor – a loop is formed in the DNA, preventing RNA polymerase from transcribing araBAD genes
Slide 69: Arabinose operon (araBAD operon)
Arabinose operon (araBAD operon) With arabinose, AraC undergoes a conformational change, allowing RNA pol to transcribe the araBAD genes ON arabinose RNA polymerase
Slide 70: Once arabinose is metabolized, switch is off
Once arabinose is metabolized, switch is off RNA polymerase OFF AraC Without arabinose, AraC acts as a repressor – a loop is formed in the DNA, preventing RNA polymerase from transcribing araBAD genes
Slide 71: Control of GFP expression - pGLO plasmid
Control of GFP expression - pGLO plasmid RNA polymerase OFF AraC Without arabinose, RNA polymerase is blocked from transcribing the GFP gene.
Slide 72: Control of GFP expression - pGLO plasmid
Control of GFP expression - pGLO plasmid With arabinose, RNA polymerase can transcribe the GFP gene ON arabinose RNA polymerase
Slide 73: pGLO plasmid
pGLO plasmid araC gene encodes the AraC repressor protein ori origin of replication pGLO pBAD promoter allows bacteria to landing site for RNA polymerase make copies of to transcribe downstream genes plasmid ampr (or bla) gfp gene gene encodes Green encodes beta-lactamase, Fluorescent Protein an enz
Slide 74: How do we produce drugs like insulin?
How do we produce drugs like insulin? 1 Clone human insulin 2 Transform 3 Select transformed 4 Culture bacteria and gene into plasmid bacteria with bacteria induce gene expression plasmid Purify protein Analyze purification 5 Lyse cells 6 7 8 Assay protein activity with with SDS-PAGE chromatography
Slide 75: Control of gene expression
Control of gene expression
Slide 76: Control of gene expression
Control of gene expression
Slide 77: Student Lab Protocol – Day 2
Student Lab Protocol – Day 2 Expression of GFP
Slide 78: Design experiment to induce GFP expression
Design experiment to induce GFP expression
Slide 79: Design experiment to induce GFP expression
Design experiment to induce GFP expression
Slide 80: Activity: Predict results after Day 2
Activity: Predict results after Day 2
Slide 81: Results & Analysis After Day 2
Results & Analysis After Day 2
Slide 82: Results after Day 2
Results after Day 2 • Why do some bacteria glow green and others do not? • Write a claim about how arabinose influenced the expression of GFP. • Revise your model.
Slide 83: Results after adding arabinose – UV
Results after adding arabinose – UV - + - + LB LB/amp
Slide 84: Small white colonies around green colonies?
Small white colonies around green colonies? + - LB/amp
Slide 85: β-lactamase diffused across plate to - side
β-lactamase diffused across plate to - side
Slide 86: Model Recombinant GFP Bacteria
Model Recombinant GFP Bacteria Day before transformation lab
Slide 87: Model Recombinant GFP Bacteria activity
Group 1 E. coli E. coli Use these images to design a model for how scientists made recombinant GFP bacteria. You can resize, copy and paste, expressin recolor, or create your own elements. Include a written g GFP description of what is happening in your model by typing over these instructions. GFP p
Slide 88: Predict Transformation Results
Predict Transformation Results On Day 1
Slide 89: Group 1
Group 1 Predict and describe the results after you do the transformation. The simplified pGLO plasmid (added to the +pGLO sides) is shown below for reference.
Slide 90: Predict Transformation Results
Predict Transformation Results After Day 2
Slide 91: Group 1
Group 1 How would you revise your model after you add arabinose? You can move or select individual colonies by double-clicking to select one and pasting it wherever you’d like.
Slide 92: Resources
Resources GLO Plasmid Map and Resources Bacterial Transformation GLO Bacterial Transformation Powerpoint err 1606) (- va Ne PGLO Plasmid @00 % How To Perform a Bactera Transformation video *
Slide 93: pGLO Resource Webpage
pGLO Resource Webpage www.bio-rad.com/teachpglo
Slide 94: Manuals
Manuals bio-rad.com/pglogenbiokit
Slide 95: pGLO Transformation Kit for General Biology 17006991EDU
pGLO Transformation Kit for General Biology 17006991EDU • 12 workstations • 2 lab activities • 3-5 lab periods
Slide 96: www.bio-rad.com/classroomresources
www.bio-rad.com/classroomresources
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