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TEACHER GUIDE: Bacterial Transformation with Mystery DNA

This teacher guide is provided to give sample answers to questions. Most of the questions are open-ended, so students may have correct answers that aren't included in this guide. Finally, although the experiment is set up to yield one correct answer, there are variations in data between students. As long as students examine their data carefully and can justify their answers based on their data, that's science! Data are always right and there isn't necessarily a 'right answer'.

Some questions to get you thinking about today’s lab:

What can we use DNA for?

We can use DNA to code for proteins, to identify individuals (like when solving a crime), or to do genetic engineering by inserting foreign DNA into an organism. 
 
How can DNA be put into bacteria?

There are three strategies for getting DNA into bacteria, which you may or may not want to talk about with students. Bacteria can insert DNA into each other by CONJUGATION; viruses can insert DNA into bacteria by TRANSDUCTION; or we can insert DNA into bacteria using chemicals or electricity, which is called TRANSFORMATION. During this lab, we will 'poke holes' in the bacteria using chemicals, allowing the DNA to flow into the bacteria- this is called BACTERIAL TRANSFORMATION.  
 
Why would we want to put DNA into bacteria?

We can use bacteria as little 'factories' to make more DNA, as they replicate, or to make protein, by transforming them with genes for proteins we want to make (like insulin). 
 
How can we tell DNA is in the bacteria once we put it there?

The DNA we insert is shaped in a little circle, called a plasmid. We can put one, two, or more genes in a single plasmid. One of the genes in the plasmid codes for the ampicillin resistance protein, and thus will allow bacteria with the plasmid DNA to grow in the presence of ampicillin. 
 
What is a plasmid? What is ampicillin?

A plasmid is a small circle of DNA. Ampicillin is an antibiotic; antibiotics prevent bacteria from growing. Ampicillin specifically prevents bacteria from making cell walls. Thus, ampicillin will not kill bacteria (that already have a cell wall), but will prevent bacteria from reproducing (because they can't make new cell walls). 
 
Materials for each group (students should work in groups of 4):

Materials to share:
Water bath at 42°C
ice
trash containers/biohazard waste bag
UV lights
 
Protocol:
1. Pipette the DNA solution from your numbered DNA tube into your E. coli bacteria tube and label the tube 1 or 2

Be sure the students number the top of the tube with which DNA they added to the bacteria.

2. Put tubes on ice for 5 minutes. Why do you think we put the tubes on ice?

To get the DNA into the bacteria, we have to poke holes in them with the chemical calcium chloride (CaCl2). CaCl2 will dissociate into Ca2+ and 2 Cl-, and the positive charge of the Ca2+ cancels the negative charge of the DNA, allowing it to cross the cell wall and cell membrane. The holes poked to allow the DNA in leaves the bacteria leaky. If we don't keep them on ice, they'll 'bleed' to death.

 
3. In the meantime, each group should get one LB agar plate and one LB agar + ampicillin plate. Write your initials and your section number on your plates. You will be plating bacteria with DNA on an LB agar plate and on an LB agar +ampicillin plate. Mark these two plates with the DNA number on your tube. Where is the best place to label your plates? What is the control you are conducting?

Always label plates on the bottom. The lids can get mixed up accidentally, so if the bottoms are labeled, the label will stay with the bacteria (which are growing in the bottom). The LB agar plate will look for the existance of viable bacteria cells. If nothing grows on the LB agar plate then your bacteria are dead and you cannot expect transformation or growth on the LB agar + ampicillin
 
4. Put tubes directly from ice into 42°C water bath for 50 seconds. What do you think heating the tubes does?

Heating the bacteria helps the holes seal shut. It's like giving the bacteria a fever, so they start to heal themselves. It's called heat shock.
 
5. Put DNA tubes directly from water bath onto ice for 2 minutes.

 
6. With a large transfer pipet, add 0.25 ml LB broth into your DNA tube ( bring solution to the first mark on the pipet.) Incubate at room temperature for 10 minutes. What is the LB broth for?

The LB (Luria-Bertani) broth is both food and water for the bacteria. It will help make the bacteria healthy after poking holes in them, shoving DNA into them, and giving them a 'fever' to help them heal.

 
7. With the small transfer pipet, pipet 0.15 ml (about half) from your DNA tube onto your LB agar plate and 0.15 ml onto your LB agar + ampicillin plate.

 
8. Spread the solutions on the plates using a sterile Q-tip. Be careful not to stab the agar.

The same Q-tip can be used for both plates as long as it is kept sterile (don't touch it to anything!).

 
9. Put your plates in a 37°C incubator for 24 hours. Why 37°C?

These bacteria are E. coli, which grow in human intestine. Because they grow in humans, they will grow best at human body temperature (37°C). You could ask students to calculate what 37°C is in Fahrenheit, or what 98.6°F is in Celsius.
 
Challenge for Day 2: What is ampicillin and why do you think we used it in some of the plates?

Ampicillin is an antibiotic (described above). We use it in some of the plates to see if the plasmid DNA is there (bacteria will only grow on ampicillin if they have the ampicillin resistance gene). We leave it out of some of the plates to make sure the bacteria can grow if there is no ampicillin present (control).
 
What do you expect to grow on each of the plates?
 

 
 
       
 
Day 2:

What do you see on your plates?

Students should look at their plates to see what they look like. They may see any of three things:
- little white dots, called colonies (each colony starts as a single bacterium because they reproduce
asexually, so each colony is like a house with a family of related bacteria)
- a big smear, called a 'bacterial lawn' (this is like a city of bacterial houses, where there are so many
colonies that we can't tell them apart any more)
- nothing, where no bacteria are growing (ampicillin may kill all of the bacteria or the students may not
have spread their bacteria around the plate correctly, e.g. they may have put it on the lid-
don't laugh! it happens!)
 
 
Now look at your plates with UV light. What do you see?



 
 
Fill in the table with your data and the class data-
what does each group (#1 and 2) see on each type of plate?

 
 
What does each DNA type (1 and 2) allow the bacteria to do?
 
#1 allows the bacteria to GROW and GLOW
#2 allows the bacteria to GROW

What would the bacteria do on each type of plate (LB agar and LB agar + ampicillin) if you added no DNA?

The bacteria would form a lawn on the LB agar plate. On the plate with ampicillin, the bacteria wouldn't grow or glow (you would see nothing) because they wouldn't have either the ampicillin resistance gene or the Green Fluorescent Protein (GFP) gene. 

After the students fill in their data tables, I usually talk about the results with them in this order.

I ask the question and help them brainstorm the answers.

After this discussion, students should be able to tell what each DNA type allows the bacteria to do.

1. Where do the bacteria grow best? On the LB agar plates, because it provides them with food, water, and shelter. Also, the plates were stored at 37°C, which is their favorite temperature.

2. If the bacteria can grow on LB agar so well, why didn't they grow on LB agar with ampicillin? This is an opportunity to talk about what ampicillin is. The bacteria aren't growing on LB agar with ampicillin because ampicillin is an antibiotic.

3. If ampicillin is an antibiotic, why doesn't it completely stop the bacteria from growing? Because of the DNA we added, the bacteria are now resistant. In fact, the DNA we added is called the 'ampicillin resistance gene'.

4. Do both DNA types have the ampicillin resistance gene? They both should grow, thus they both have the ampicillin resistance gene.

5. What would happen if no DNA is added? The bacteria could not grow in the presence of ampicillin if they do not contain the ampicillin resistance gene.

6. #1 DNA contains a gene that codes for Green Fluorescent Protein (GFP) , which is why #1 bacteria GLOW. What are #1 bacteria able to do? GLOW AND GROW. What are #2 bacteria able to do? GROW.

7. Why do you want to do this kind of GENETIC ENGINEERING experiment? Say you know someone who is diabetic. They have to take the protein insulin to be healthy. We can put the insulin gene into a plasmid and then insert that plasmid into bacteria. These bacteria will make insulin for diabetics to use. Before genetic engineering was invented, we used to have to kill pigs to get their insulin. Now we can use bacteria to make human insulin (instead of using pig insulin, especially important if someone is allergic to pigs) and we don't have to kill any animals to do it.

BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona Last Modified March 8, 2002 Nadja Anderson, Ph.D. nadjal@email.arizona.edu http://biotech.biology.arizona.edu