pGLO Bacterial Transformation

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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.

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© 2020 Bio-Rad Laboratories, Inc.

Why genetically modify organisms?

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• Disease/drought/pest resistance.
• Increased nutrition

• Modified animal models for research
• Cancer, obesity, heart disease, etc.

• Modified mosquitoes to fight disease

• Drug production like insulin, hormones, vaccines, and anti-cancer drugs.

Brief history of insulin

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• 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 engineered E. coli (recombinant DNA, or rDNA).
• 1982 – Humulin approved by the FDA.

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The protein products of biotech

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How can we make LOTS of protein?

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1. Identify a gene for a protein.

Gene

How can we make LOTS of protein?

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1. Identify a gene for a protein.
2. Put the gene into bacteria.

E. coli

How can we make LOTS of protein?

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1. Identify a gene for a protein.
2. Put the gene into bacteria.
3. Grow lots of the bacteria.

How can we make LOTS of protein?

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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!

DNA

mRNA

protein

How can we make LOTS of protein?

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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!

How can we make LOTS of protein?

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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.

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How do you get genes into bacteria?

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1. Make a plasmid with your gene.
2. Do bacterial transformation. This is what you’ll do in this activity.

E. coli

Plasmid

Genetic engineering using plasmids

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• Bacteria often have plasmids — circular loops of DNA
• Bacteria can also take in new plasmids.

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Bacteria

Chromosome

Plasmids

Genetic engineering using plasmids

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• Scientists can modify or engineer plasmids for specific purposes.

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Antibiotic resistance�Allows transformed bacteria to survive on plates with antibiotic

Origin of replication�Let’s the bacteria make copies of the plasmid

Genes of interest�Genes for protein production or other desired trait

Green fluorescent protein

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Under visible light

Under ultraviolet (UV) light

The jellyfish Aequorea Victoria has a gene for green fluorescent protein which glows green under UV light.

pGLO plasmid

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• The pGLO plasmid is engineered to have the GFP gene from Aequorea victoria.

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Beta-lactamase�Allows transformed bacteria to survive on plates with ampicillin

araC�Gene for the protein AraC that controls the GFP gene like and ON/OFF switch.

GFP�Gene for green fluorescent protein.

pGLO

ori

pGLO plasmid DNA

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Bacterial Membrane

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E. coli

Plasma Membrane

Non-polar

Polar

Add plasmid

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E. coli

Plasmid DNA

Plasma Membrane

Add plasmid

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E. coli

Negative charges on DNA backbone

Add transformation solution (CaCl₂)

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E. coli

Ca²⁺ shields charges on DNA to make it less polar

Heat Shock

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E. coli

Add heat to create pores in the membrane

Heat Shock

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E. coli

Add heat to create pores in the membrane

Heat Shock

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E. coli

Plasmid enters cell through pore

Recovery on ice, 2 min

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E. coli

Pores close

Add LB broth, allow gene expression, 10 min

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E. coli

Add LB broth, allow gene expression, 10 min

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E. coli

AraC, regulatory protein

GFP, only if arabinose is in the media

Beta-lactamase, ampicillin resistance

Selective media – ampicillin

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Selective media – ampicillin

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Beta-lactamase, ampicillin resistance

Selective media – ampicillin

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Ampicillin on the plate

Bacteria without the plasmid cannot grow in the presence of ampicillin

Selective media – ampicillin

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• 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.

LB Broth

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• 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

Transformation summary

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Label tubes

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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.

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Pick colonies

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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.

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Add plasmid DNA

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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.

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10 min

Label plates

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10. While your tubes are on ice, label the bottom of your plates.
11. Add your group ID or initials.

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LB

–pGLO

LB/amp

–pGLO

LB/amp

+pGLO

LB/amp/ara

+pGLO

Heat shock

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Get your timers ready!

12. Heat shock tubes at 42°C for exactly 50 sec.
13. Immediately return tubes to ice for 2 min.

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• Add 250 µl LB broth to both tubes.
• Leave at room temperature for 10 min.

Meanwhile…

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E. coli

Plasmid genes are expressed

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E. coli

Beta-lactamase�

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E. coli

Beta-lactamase, ampicillin resistance

Beta-lactamase makes E. coli resistant to ampicillin

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• 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.

AraC�

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E. coli

AraC, regulatory protein

AraC Controls Expression of GFP

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• Arabinose �

AraC

• Without arabinose, the switch is OFF. AraC blocks RNA polymerase , and the GFP gene is not transcribed.�

• With arabinose , the switch is ON. AraC changes shape and RNA transcribes the GFP gene.�

RNA polymerase

Green Fluorescent Protein (GFP)

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E. coli

GFP, only if arabinose is in the media

Plating Bacteria

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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.

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Transformation Efficiency

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• How successful was your transformation?�You can calculate the transformation efficiency and compare with other groups.

87 colonies growing on plate

0.16 μg of DNA spread

= 543 transformants/μg

(or 5.4 x 10² transformants/μg)

Example

Extra graphics

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