TRANSFORMATION

Intro to Lab #8

Figure 20.2 Bacterium

Bacterialchromosome

Plasmid

2

1

3

4

Gene inserted intoplasmid

Cell containing geneof interest

RecombinantDNA (plasmid)

Gene of interest

Plasmid put intobacterial cell

DNA ofchromosome(“foreign” DNA)

Recombinantbacterium

Host cell grown in culture toform a clone of cells containingthe “cloned” gene of interest

Gene of interest

Protein expressed fromgene of interest

Protein harvestedCopies of gene

Basic researchand variousapplications

Basicresearchon protein

Basic research on gene

Gene for pestresistance insertedinto plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growthhormone treatsstunted growth

Transformation1. Identify a restriction enzyme that will

cut out gene of interest and cut open the plasmid

2. Isolate DNA from 2 sources (making sure the at the plasmid has a genetic marker)

3. Cut both types of DNA with the same restriction enzyme

4. Mix the 2 types of DNA to join them through complementary base pairing

5. Add DNA ligase to bond DNA covalently producing Recombinant DNA

6. Incubate bacteria at 42 C with calcium chloride; bacteria become competent / permeable - so that the bacteria will take in the plasmid (TRANSFORMATION)

7. Use a genetic marker to identify bacteria with the recombinant plasmid

8. Clone bacteria

Pre-Lab Inquiry

You work on a team in a research lab. You have three separate plasmids in your lab, but the labels have come off of the tubes and you no longer know which plasmid is which. For the time being, you will refer to the plasmids are plasmids 1, 2, and 3. You know the following: One plasmid has kanamycin-resistant gene. Two plasmids have an ampicillin-resistance gene. In addition to having an antibiotic-resistance

marker, one plasmid also codes for the gene for green fluorescent protein (GFP), a protein from the bioluminescent jellyfish Aequorea victoria.

Pre-lab Inquiry

Bacteria that produce the green fluorescent protein look green under white light and fluoresce under ultraviolet light

Assume that each research team has access to the following materials and is responsible for identifying one of the three plasmid. (Note: you will not actually use any materials during this pre-lab inquiry). Before planning your experiment, answer the following questions to ensure that you understand some of the basic concepts involved in the lab. Some of these questions are specifically designed to help you think about setting up your experiment.

Materials Available

Starter plates (containing E. coli in rapid growth)

Kanamycin plates Ampicillin plates LB plates (plates contain Luria Broth/no

antibiotic) Plasmids 1, 2, and 3 Transformation agents, tubes, and

equipment (inoculating loops, calcium chloride, ice, 42C water bath, incubator)

Questions

1. What is a plasmid? Small, usually circular, extra-chromosomal

piece of DNA that exits in nature in some bacteria and yeasts. They can be transferred between organisms. In the lab they can be used to manipulate and introduce DNA of interest into bacterium.

2. What is transformation? The uptake of exongenous, naked DNA by a

cell. The newly adopted DNA can become a heritable part of the cell’s genetic material.

Questions

3. Why is naturally occurring transformation beneficial to bacteria? It provides them with access to new genetic

material, which increases their chances of being able to adapt to the environment.

4. Why is transformation useful to research scientists? It enables them to introduce foreign DNA into

bacteria. This allows them to further study and work with genes on the plasmid DNA and the proteins that the genes code for.

Questions

5. Should you plate some of your transformed bacteria onto plates with antibiotic? Why or Why not? Yes – you will select for/isolate bacteria that have been

transformed with the plasmid with the resistance gene 6. What would you expect to see if you plated

some of your transformed bacteria onto a plate without antibiotic? Would there be an advantage to doing this? Explain. You would see a lawn of bacteria (the plate would be

covered). This tells you that the bacteria survived the transformation process. When compared to the antibiotic plates it gives an indication of how many bacteria were transformed

Questions

7. To transform bacteria with plasmids, technicians first make the bacteria competent (capable of taking up DNA) by placing them in calcium chloride and chilling them. Plasmid is then added to the competent bacteria and the plasmid/bacteria combination is taken through a few more steps to make the bacteria take up DNA. In your experiment, should you treat a tube of bacteria that you do not add plasmid to exactly as you do the tube of bacteria that you will transform? Why or why not? This provides a control demonstrating that the antibiotic-

resistance colonies that appear on the plate are a result of the plasmid being taken up by the cells. If no plasmid, then there should be no growth on antibiotic plates.

What would you expect?

LB AMP plates LB Kan plates LB plates

plasmid

+ plmd

- plmd + plmd

- plmd

+ plmd

- plmd Color when

present

Amp

Kan

Amp & GFP

What would you expect?

LB AMP plates LB Kan plates LB plates

plasmid

+ plmd

- plmd + plmd

- plmd

+ plmd

- plmd Color when

present

Amp 25 -100

colonies

0 colonie

s

0colonie

s

0 colonie

s

Lawn Lawn yellow

Kan 0 colonie

s

0 colonie

s

25-100 colonie

s

0 colonie

s

Lawn Lawn Yellow

Amp & GFP

25-100 colonie

s

0 colonie

s

0 colonie

s

0 colonie

s

Lawn Lawn Yellow/green or

green

What you need…

2 LB plates 2 LB/Kan plates 2 LB/Amp plates 2 sterile transformation tubes & rack Container with crushed ice 3 sterile inoculating loops 6 sterile transfer pipets Waste container 3 mL vial of LB 3 mL vial of CaCl2 (on ice)

Basic Outline of the Lab

1. Label 1 tube + / 1 tube –

2. Add CaCl2, place on ice

3. Use sterile loop to transfer E. coli to both tubes (no agar!) return to ice

4. Use sterile loop to add loopful of plasmid (1, 2, or 3) to + tube

5. Ice for 15 min & label 1of each plate “+” and 1 of each “-”

6. Heat shock (42C for 90 sec)

7. Add luria broth & sit at room temp 5 to 15 minutes

8. Spread bacteria on plates