Bio-rad Laboratories, Inc. · NYSE:BIO
Bio-rad Laboratories — Technical Presentation (2023)
80 slides from Bio-rad Laboratories's technical presentation, 2023
Slide 1: pGLO Bacterial Transformation
pGLO Bacterial Transformation 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. bio-rad.com/explorer
Slide 2: Slide Deck Overview
Slide Deck Overview Background Student Results and Information Lab Protocol Analysis Genetic engineering, GFP, plasmids , and bacterial transformation Antibiotic selection Control of gene expression with a rabinose operon Resources Bacterial transformation steps
Slide 3: Background Information
Background Information Genetic engineering, GFP, plasmids, and bacterial transformation
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: Cloning a gene
Cloning a gene @ a~ yO, O
Slide 21: 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 – plant, animal, human, bacterial.
Slide 22: Bacteria divide, and new bacteria get plasmids
Bacteria divide, and new bacteria get plasmids
Slide 23: Genes are transcribed and translated
Genes are transcribed and translated transcription translation protein
Slide 24: Activity: Model recombinant GFP bacteria
Activity: Model recombinant GFP bacteria
Slide 25: Background Information: Antibiotic selection
Background Information Antibiotic selection
Slide 26: 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 27: Selection of transformed bacteria
Selection of transformed bacteria
Slide 28: Plasmids can carry multiple genes
Plasmids can carry multiple genes
Slide 29: Selection of transformed bacteria
Selection of transformed bacteria
Slide 30: 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 31: Transformation with plasmid with ampr
Transformation with plasmid with ampr
Slide 32: β-lactamase produced
β-lactamase produced
Slide 33: Bacteria plated on ampicillin plates
Bacteria plated on ampicillin plates
Slide 34: β-lactamase breaks down ampicillin
β-lactamase breaks down ampicillin
Slide 35: Bacteria that aren’t transformed don’t grow
Bacteria that aren’t transformed don’t grow
Slide 36: Transformed bacteria grow into colonies
Transformed bacteria grow into colonies
Slide 37: Background Information: Control of gene expression with arabinose operon
Background Information Control of gene expression with arabinose operon
Slide 38: 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 39: Without arabinose in the media
Without arabinose in the media OFF
Slide 40: With arabinose in the media
With arabinose in the media ON
Slide 41: 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 42: 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 43: 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 44: 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 45: 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 46: 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 47: 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 48: 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 49: Control of gene expression
Control of gene expression
Slide 50: Control of gene expression
Control of gene expression
Slide 51: Background Information: Bacterial transformation steps
Background Information Bacterial transformation steps
Slide 52: DNA is negatively charged
DNA is negatively charged
Slide 53: CaCl2 transformation solution
CaCl2 transformation solution negative charges plasmid plasma membran e
Slide 54: CaCl2 transformation solution
CaCl2 transformation solution Ca2+ shields negative charges
Slide 55: Incubate on ice for 10 min
Incubate on ice for 10 min Ca2+ shields negative charges
Slide 56: Heat shock at 42°C for 50 sec
Heat shock at 42°C for 50 sec
Slide 57: Incubate on ice for 2 min
Incubate on ice for 2 min
Slide 58: 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 59: 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 60: Student Lab Protocol
Student Lab Protocol Bacterial transformation with pGLO plasmid
Slide 61: Lab protocol
Lab protocol 1. Label 1 empty tube – and the other +. Place tubes in foam rack. 250 µl 2. Add 250 μl of transformation solution (TS) into each tube. TS – + 3. Place both tubes on ice. transformatio n solution ice
Slide 62: Lab protocol
Lab protocol 4. Use a loop to pick up 2-4 large colonies of bacteria from the starter plate. 5. Put the loop into the transformation solution in the – tube. Spin the loop between your fingers about 30 seconds, until the bacteria is completely – + distributed (no chunks). Close the tube and put it ba
Slide 63: 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 64: Lab protocol
Lab protocol 7. Use a new loop to transfer a loopful (~10 μl) of pGLO plasmid to the + tube. Swirl the loop in the tube to mix. Close the tube and put back on ice. – + Important! Do NOT put pGLO plasmid into – tube! 8. Incubate both tubes on ice for 10 minutes. ice 10m
Slide 65: Lab protocol
Lab protocol 9. While the tubes are on ice, label agar plates: • LB plate: – • LB/amp plate: – • LB/amp plate: + • LB/amp/ara plate:+
Slide 66: Lab protocol
Lab protocol 10. Heat shock. Bring your tubes on ice to the water bath. Set time for 50 seconds. Place the rack with the tubes in the 10m 50s 2m 42°C water bath for exactly 50 seconds. 11. Put tubes on ice and incubate for 2 minutes. 42°C
Slide 67: Lab protocol
Lab protocol 12. Move the tubes out of the ice and onto the benchtop. 13. Add 250 μl of LB broth (LB) to each tube. Use a new pipet for each tube. 14. Close the tubes and incubate at room temperature for 10 LB minutes. – + 10m
Slide 68: Lab protocol
Lab protocol 15. Flick the tubes a few times to mix up the bacteria. 100 µl 16. Add 100 μl (about 1 drop) of bacteria from each tube to the appropriate plates. Use a new – pipet for each tube. . +
Slide 69: Lab protocol
Lab protocol 17. Use a loop to spread the bacteria all over the agar. Use a new loop for each plate. 18. Stack plates upside down, tape together, and add lab group or initials. Incubate at 37°C overnight, or at room temperature for 2 days.
Slide 70: Results & Analysis
Results & Analysis After Day 1
Slide 71: Predict results
Predict results LB LB/amp LB/amp LB/amp/ – – + ara + Ara
Slide 72: Expected results
Expected results LB LB/amp LB/amp LB/amp/ – – + ara + Off-white lawn No bacterial Many off-white Many off-white of bacteria growth colonies (~75) colonies that covering plate glow green under UV light
Slide 73: Calculate transformation efficiency
Calculate transformation efficiency Exampl e 87 colonies growing on plate = 543 transformants / μg 0.16 μg of DNA spread (or 5.4 x 102 transformants / μg)
Slide 74: Model Recombinant GFP Bacteria
Model Recombinant GFP Bacteria Day before transformation lab
Slide 75: 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 76: 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 77: pGLO Resource Webpage
pGLO Resource Webpage www.bio-rad.com/teachpglo
Slide 78: Instructor manual and student quick guide
Instructor manual and student quick guide
Slide 79: pGLO Bacterial Transformation Kit 1660003EDU
pGLO Bacterial Transformation Kit 1660003EDU • 8 workstations • 1 lab activity • 2 lab periods
Slide 80: www.bio-rad.com/classroomresources
www.bio-rad.com/classroomresources