1 of 43

pGLO Bacterial Transformation

Student presentation for use with �the pGLO Bacterial Transformation Kit

explorer.bio-rad.com

BIO-RAD is a trademark of Bio-Rad Laboratories, Inc. All trademarks used herein are the property of their respective owner.

2 of 43

Why genetically modify organisms?

explorer.bio-rad.com

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.

3 of 43

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

explorer.bio-rad.com

4 of 43

The protein products of biotech

explorer.bio-rad.com

	Used to treat	Made in	Price per gram
Gold	N/A	N/A	$40
Insulin	Diabetes	E. coli	$60
Human Growth Hormone	Growth disorders	E. coli	$227,000
Granulocyte Colony Stimulating Factor	Cancers	E. coli	$1,357,000

5 of 43

How can we make LOTS of protein?

Identify a gene for a protein.

explorer.bio-rad.com

Gene

6 of 43

How can we make LOTS of protein?

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

explorer.bio-rad.com

E. coli

7 of 43

How can we make LOTS of protein?

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

explorer.bio-rad.com

8 of 43

How can we make LOTS of protein?

Identify a gene for a protein.
Put the gene into bacteria.
Grow lots of the bacteria.
The bacteria transcribe and translate the gene — mini protein factories!

explorer.bio-rad.com

DNA

mRNA

protein

9 of 43

How can we make LOTS of protein?

Identify a gene for a protein.
Put the gene into bacteria.
Grow lots of the bacteria.
The bacteria transcribe and translate the gene — mini protein factories!

explorer.bio-rad.com

10 of 43

How can we make LOTS of protein?

Identify a gene for a protein.
Put the gene into bacteria.
Grow lots of the bacteria.
The bacteria transcribe and translate the gene — mini protein factories!
Purify the protein.

explorer.bio-rad.com

11 of 43

How do you get genes into bacteria?

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

explorer.bio-rad.com

E. coli

Plasmid

12 of 43

Genetic engineering using plasmids

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

explorer.bio-rad.com

Bacteria

Chromosome

Plasmids

13 of 43

Genetic engineering using plasmids

Scientists can modify or engineer plasmids for specific purposes.

explorer.bio-rad.com

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

14 of 43

Green fluorescent protein

explorer.bio-rad.com

Under visible light

Under ultraviolet (UV) light

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

15 of 43

pGLO plasmid

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

explorer.bio-rad.com

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

16 of 43

pGLO plasmid DNA

explorer.bio-rad.com

17 of 43

Bacterial Membrane

explorer.bio-rad.com

E. coli

Plasma Membrane

Non-polar

Polar

18 of 43

Add plasmid

explorer.bio-rad.com

E. coli

Plasmid DNA

Plasma Membrane

19 of 43

Add plasmid

explorer.bio-rad.com

E. coli

Negative charges on DNA backbone

20 of 43

Add transformation solution (CaCl₂)

explorer.bio-rad.com

E. coli

Ca²⁺ shields charges on DNA to make it less polar

21 of 43

Heat Shock

explorer.bio-rad.com

E. coli

Add heat to create pores in the membrane

22 of 43

Heat Shock

explorer.bio-rad.com

E. coli

Add heat to create pores in the membrane

23 of 43

Heat Shock

explorer.bio-rad.com

E. coli

Plasmid enters cell through pore

24 of 43

Recovery on ice, 2 min

explorer.bio-rad.com

E. coli

Pores close

25 of 43

Add LB broth, allow gene expression, 10 min

explorer.bio-rad.com

E. coli

26 of 43

Add LB broth, allow gene expression, 10 min

explorer.bio-rad.com

E. coli

AraC, regulatory protein

GFP, only if arabinose is in the media

Beta-lactamase, ampicillin resistance

27 of 43

Selective media – ampicillin

explorer.bio-rad.com

28 of 43

Selective media – ampicillin

explorer.bio-rad.com

Beta-lactamase, ampicillin resistance

29 of 43

Selective media – ampicillin

explorer.bio-rad.com

Ampicillin on the plate

Bacteria without the plasmid cannot grow in the presence of ampicillin

30 of 43

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

31 of 43

AraC Controls Expression of GFP

Arabinose �(a sugar) works like a switch.

explorer.bio-rad.com

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

32 of 43

Green Fluorescent Protein (GFP)

explorer.bio-rad.com

E. coli

GFP, only if arabinose is in the media

33 of 43

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

explorer.bio-rad.com

34 of 43

Transformation summary

explorer.bio-rad.com

1.	CaCl₂ transformation solution	Shields negative charge on DNA.
2.	Pre-heat shock incubation on ice	Slows fluid plasma membrane for greater shock.
3.	Heat shock	Increases permeability of cell membranes.
4.	Post-heat shock incubation on ice	Restores cell membrane.
5.	Incubation at room temperature with LB broth	Allows beta-lactamase expression so bacteria can grow on plates with ampicillin.
6.	Spread on LB/amp plates	Selects for transformed bacteria and allows formation of colonies.

35 of 43

Label tubes

You have tubes with 250 µl transformation solution.

Label one +pGLO and the other –pGLO.
Add your initials.
Place into foam rack and on ice.

explorer.bio-rad.com

36 of 43

Pick colonies

Using a sterile loop�pick 1–2 large E. coli colonies.
Add to the +pGLO tube. Spin the loop to disperse the bacteria. No clumps!
Using a new loop, at 1–2 colonies to –pGLO tube.
Place tubes into foam rack and on ice.

explorer.bio-rad.com

37 of 43

Add plasmid DNA

Add 10 µl (1 loop full) pGLO plasmid to +pGLO tube.�DO NOT ADD TO –pGLO tube.
Place tubes into foam rack and on ice for 10 min.

explorer.bio-rad.com

10 min

38 of 43

Label plates

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

explorer.bio-rad.com

–pGLO

LB/amp

–pGLO

LB/amp

+pGLO

LB/amp/ara

+pGLO

39 of 43

Heat shock

Get your timers ready!

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

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

explorer.bio-rad.com

40 of 43

Plating Bacteria

Flick tubes to mix.
Using a new sterile pipet, add 100 µl bacteria to appropriate plates (+pGLO or –pGLO).
Use a loop to spread bacteria evenly.�Use a new loop for each plate.
Incubate overnight at 37°C or for 2 days at room temperature.

explorer.bio-rad.com

41 of 43

explorer.bio-rad.com

		Plates
		–pGLO LB	–pGLO LB/amp	+pGLO LB/amp	+pGLO LB/amp/ara
Components	Bacteria
	DNA
	Ampicillin
	Arabinose
	Grow?
	Glow?

42 of 43

Transformation Efficiency

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

explorer.bio-rad.com

87 colonies growing on plate

0.16 μg of DNA spread

= 543 transformants/μg

(or 5.4 x 10² transformants/μg)

Example

1 of 43

2 of 43

3 of 43

4 of 43

5 of 43

6 of 43

7 of 43

8 of 43

9 of 43

10 of 43

11 of 43

12 of 43

13 of 43

14 of 43

15 of 43

16 of 43

17 of 43

18 of 43

19 of 43

20 of 43

21 of 43

22 of 43

23 of 43

24 of 43

25 of 43

26 of 43

27 of 43

28 of 43

29 of 43

30 of 43

31 of 43

32 of 43

33 of 43

34 of 43

35 of 43

36 of 43

37 of 43

38 of 43

39 of 43

40 of 43

41 of 43

42 of 43

43 of 43