Bio-Rad Laboratories, Inc. · NYSE:BIO
Bio-Rad Laboratories — Technical Presentation (2020)
47 slides from Bio-Rad Laboratories's technical presentation, 2020
Slide 1: pGLO Bacterial Transformation
pGLO Bacterial Transformation Student presentation for use with the pGLO Bacterial Transformation Kit BIO-RAD is a trademark of Bio-Rad Laboratories, Inc. All trademarks used herein are the property of their respective owner. explorer.bio-rad.com © 2020 Bio-Rad Laboratories, Inc.
Slide 2: Why genetically modify organisms?
Why genetically modify organisms? • Modified animal models for research • Cancer, obesity, heart • Modified mosquitoes to disease, etc. fight disease • Drug production like insulin, • Disease/drought/pest hormones, vaccines, and resistance. anti-cancer drugs. • Increased nutrition explorer.bio-rad.c
Slide 3: Brief history of insulin
Brief history of insulin • 1922 – Canadian researchers isolate insulin, cure diabetics using bovine insulin, and win the Nobel Prize in 1923. Previously, diabetes had been a virtual death sentence – there was no treatment • 1978 – scientists at Genentech produce human insulin using genetically engin
Slide 4: The protein products of biotech
The protein products of biotech Used to treat Made in Price per gram Gold N/A N/A $40 Insulin Diabetes E. coli $60 Human Growth Growth disorders E. coli $227,000 Hormone Granulocyte Colony Cancers E. coli $1,357,000 Stimulating Factor explorer.bio-rad.com
Slide 5: How can we make LOTS of protein?
How can we make LOTS of protein? 1. Identify a gene for a Gene protein. explorer.bio-rad.com
Slide 6: How can we make LOTS of protein?
How can we make LOTS of protein? 1. Identify a gene for a protein. 2. Put the gene into E. coli bacteria. explorer.bio-rad.com
Slide 7: How can we make LOTS of protein?
How can we make LOTS of protein? 1. Identify a gene for a protein. 2. Put the gene into bacteria. 3. Grow lots of the bacteria. explorer.bio-rad.com
Slide 8: How can we make LOTS of protein?
How can we make LOTS of protein? 1. Identify a gene for a protein. DNA 2. Put the gene into mRNA bacteria. protein 3. Grow lots of the bacteria. 4. The bacteria transcribe and translate the gene — mini protein factories! explorer.bio-rad.com
Slide 9: How can we make LOTS of protein?
How can we make LOTS of protein? 1. Identify a gene for a protein. 2. Put the gene into bacteria. 3. Grow lots of the bacteria. 4. The bacteria transcribe and translate the gene — mini protein factories! explorer.bio-rad.com
Slide 10: How can we make LOTS of protein?
How can we make LOTS of protein? 1. Identify a gene for a protein. 2. Put the gene into bacteria. 3. Grow lots of the bacteria. 4. The bacteria transcribe and translate the gene — mini protein factories! 5. Purify the protein. explorer.bio-rad.com
Slide 11: How do you get genes into bacteria?
How do you get genes into bacteria? 1. Make a plasmid with your gene. 2. Do bacterial transformation. This is what you’ll do in this activity. Plasmid E. coli explorer.bio-rad.com
Slide 12: Genetic engineering using plasmids
Genetic engineering using plasmids • Bacteria often have plasmids — circular loops of DNA • Bacteria can also take in new plasmids Chromosome Bacteria Plasmids explorer.bio-rad.com
Slide 13: Genetic engineering using plasmids
Genetic engineering using plasmids Scientists can modify or engineer plasmids for specific purposes. Origin of replication Genes of interest Let’s the bacteria make Genes for protein copies of the plasmid production or other desired trait Antibiotic resistance Allows transformed bacteria to survive
Slide 14: Green fluorescent protein (GFP)
Green fluorescent protein (GFP) The jellyfish Aequorea Victoria has a gene for green fluorescent protein which glows green under UV light. Under visible light Under ultraviolet (UV) light explorer.bio-rad.com
Slide 15: pGLO plasmid
pGLO plasmid The pGLO plasmid is engineered to have the GFP gene from Aequorea victoria. araC Gene for the protein AraC that controls the ori GFP gene like and pGLO ON/OFF switch. Beta-lactamase GFP Allows transformed Gene for green bacteria to survive on fluorescent protein. plates with ampicillin
Slide 16: pGLO plasmid DNA
pGLO plasmid DNA explorer.bio-rad.com
Slide 17: Bacterial membrane
Bacterial membrane E. coli Plasma Membrane Non-polar Polar explorer.bio-rad.com
Slide 18: Add plasmid
Add plasmid Plasmid DNA E. coli Plasma Membrane explorer.bio-rad.com
Slide 19: Add plasmid
Add plasmid Negative charges on DNA backbone E. coli explorer.bio-rad.com
Slide 20: Add transformation solution (CaCl2)
Add transformation solution (CaCl2) E. coli Ca2+ shields charges on DNA to make it less polar explorer.bio-rad.com
Slide 21: Heat shock
Heat shock E. coli Add heat to create pores in the membrane explorer.bio-rad.com
Slide 22: Heat shock
Heat shock E. coli Add heat to create pores in the membrane explorer.bio-rad.com
Slide 23: Heat shock
Heat shock Plasmid enters cell through pore E. coli explorer.bio-rad.com
Slide 24: Recovery on ice, 2 min
Recovery on ice, 2 min Pores close E. coli explorer.bio-rad.com
Slide 25: Add LB broth, allow gene expression, 10 min
Add LB broth, allow gene expression, 10 min E. coli explorer.bio-rad.com
Slide 26: Add LB broth, allow gene expression, 10 min
Add LB broth, allow gene expression, 10 min AraC, regulatory protein E. coli Beta-lactamase, GFP, only if ampicillin arabinose is resistance in the media explorer.bio-rad.com
Slide 27: Selective media – ampicillin
Selective media – ampicillin explorer.bio-rad.com
Slide 28: Selective media – ampicillin
Selective media – ampicillin Beta-lactamase, ampicillin resistance explorer.bio-rad.com
Slide 29: Selective media – ampicillin
Selective media – ampicillin Ampicillin on the plate Bacteria without the plasmid cannot grow in the presence of ampicillin explorer.bio-rad.com
Slide 30: Selective media – ampicillin
Selective media – ampicillin • Transformed bacteria (with the plasmid) will make beta- lactamase , which breaks down ampicillin. This enables them to grow on ampicillin plates • Bacteria without the plasmid (NOT transformed) cannot grow on plates with ampicillin explorer.bio-rad.com
Slide 31: 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: o Carbohydrates o Amino acids o Nucleotides o Salts o Vitamins explorer.bio-rad.com
Slide 32: Transformation summary
Transformation summary 1. CaCl2 transformation Shields negative charge on DNA solution 2. Pre-heat shock Slows fluid plasma membrane for greater incubation on ice shock 3. Heat shock Increases permeability of cell membranes 4. Post-heat shock Restores cell membrane incubation on ice 5. Incubation at
Slide 33: Label tubes
Label tubes You have tubes with 250 µl transformation solution. 1. Label one +pGLO and the other –pGLO. 2. Add your initials. 3. Place into foam rack and on ice. explorer.bio-rad.com
Slide 34: Pick colonies
Pick colonies 4. Using a sterile loop pick 1–2 large E. coli colonies. 5. Add to the +pGLO tube. Spin the loop to disperse the bacteria. No clumps! 6. Using a new loop, at 1–2 colonies to –pGLO tube. 7. Place tubes into foam rack and on ice. explorer.bio-rad.com
Slide 35: Add plasmid DNA
Add plasmid DNA 8. Add 10 µl (1 loop full) pGLO plasmid to +pGLO tube. DO NOT ADD TO –pGLO tube. 9. Place tubes into foam rack and on ice for 10 min. 10 min explorer.bio-rad.com
Slide 36: Label plates
Label plates 10. While your tubes are on ice, label the bottom of your plates. 11. Add your group ID or initials. LB LB/amp LB/amp LB/amp/ara –pGLO –pGLO +pGLO +pGLO explorer.bio-rad.com
Slide 37: Heat shock
Heat shock Get your timers ready! 12. Heat shock tubes at 42°C for exactly 50 sec. 13. Immediately return tubes to ice for 2 min. 14. Add 250 µl LB broth to both tubes. 15. Leave at room temperature for 10 min. explorer.bio-rad.com
Slide 38: Meanwhile…
Meanwhile… E. coli explorer.bio-rad.com
Slide 39: Plasmid genes are expressed
Plasmid genes are expressed E. coli explorer.bio-rad.com
Slide 40: Beta-lactamase
Beta-lactamase E. coli Beta-lactamase, ampicillin resistance explorer.bio-rad.com
Slide 41: Beta-lactamase makes E. coli resistant to ampicillin
Beta-lactamase makes E. coli resistant to ampicillin • Transformed bacteria (with the plasmid) will make beta- lactamase , which breaks down ampicillin . This enables them to grow on ampicillin plates • Bacteria without the plasmid (NOT transformed) cannot grow on plates with ampicillin explorer.bio
Slide 42: AraC
AraC AraC, regulatory protein E. coli explorer.bio-rad.com
Slide 43: AraC controls expression of GFP
AraC controls expression of GFP • Arabinose AraC (a sugar) works like a switch • Without arabinose, the switch is OFF. AraC blocks RNA RNA polymerase polymerase , and the GFP gene is not transcribed • With arabinose , the switch is ON. AraC changes shape and RNA transcribes the GFP gene explorer.bio
Slide 44: Green fluorescent protein (GFP)
Green fluorescent protein (GFP) E. coli GFP, only if arabinose is in the media explorer.bio-rad.com
Slide 45: Plating bacteria
Plating bacteria 16. Flick tubes to mix. 17. Using a new sterile pipet, add 100 µl bacteria to appropriate plates (+pGLO or –pGLO). 18. Use a loop to spread bacteria evenly. Use a new loop for each plate. 19. Incubate overnight at 37°C or for 2 days at room temperature. explorer.bio-rad.com
Slide 46: Components
Plates –pGLO –pGLO +pGLO +pGLO LB LB/amp LB/amp LB/amp/ara Bacteria Components DNA Ampicillin Arabinose Grow? Glow? explorer.bio-rad.com
Slide 47: Transformation efficiency
Transformation efficiency • How successful was your transformation? You can calculate the transformation efficiency and compare with other groups. 87 colonies growing on plate Example = 543 transformants/μg 0.16 μg of DNA spread (or 5.4 x 102 transformants/μg) explorer.bio-rad.com