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Activity 4: Transformation of E. coli using green fluorescent protein

Information for Teachers

Although the E. coli strain used in these experiments has been rendered non-pathogenic, it is important to teach the students good sterile technique and safe disposal of bacteria.

• Gloves and safety glasses are to be worn at all times during this experiment.
• Keep nose and mouth away from tip end when pipetting suspension culture to avoid inhaling any aerosol that might be created.
• Use a 10% bleach solution to wipe down the benches at the end of the experiment.
• Wash hands before leaving lab.

To dispose of contaminated material:

Immerse all disposable pipets, tubes, and loops that have come in contact with bacteria in 10% bleach solution for at least 20 minutes before draining, rinsing and disposing of in the trash.

When lab is complete, collect all petri dishes, open, and immerse in a 10% bleach solution to kill all bacteria. Allow materials to stand in bleach solution for 20 minutes or more. Drain excess solution, seal materials in a plastic bag and dispose in the trash.

Introduction

Transformation of cells is a widely used and versatile tool in genetic engineering and is of critical importance in the development of molecular biology. The purpose of this technique is to introduce a foreign plasmid into bacteria, the bacteria then amplifies the plasmid, making large quantities of it.

A plasmid is a small circular piece of DNA (about 2,000 to 10,000 base pairs) that contains important genetic information for the growth of bacteria. Bacteria, which often grow in the same environment as molds and fungi, evolved to make proteins that inactivate the toxins produced by these other organisms. The bacteria share this vital information by passing it among themselves in the form of genes in plasmids. Hence, the natural function of a plasmid is to transfer genetic information vital to the survival of the bacteria. It is this characteristic of plasmids that is exploited for use in transformation.

For bacteria to take in a plasmid, they must first be made "competent" to take up DNA, which won't normally pass through a bacterial cell's membrane. This is done by creating small holes in the bacterial cells by suspending them in a solution with a high concentration of calcium. DNA can then be forced into the cells by the procedures followed during this experiment.

In this activity, students will use a strain of E. coli that has been made competent to allow it to incorporate and express a plasmid containing two genes. One gene codes for a green fluorescent protein (GFP) and the other codes for ampicillin resistance. The source of the GFP gene is the bioluminescent jellyfish Aequorea victoria. The ampicillin-resistance gene allows us to select which of the E. coli cells have been transformed based on their ability to grow in an environment that contains the antibiotic ampicillin.

Figure 01 - Click to Enlarge

Objectives

1. Understand recombinant DNA techniques, in particular the transformation procedure using the heat shock method.
2. Understand the uses of marker or reporter genes in molecular biology experiments and how to screen for a gene of interest.
3. Understand that DNA can be transferred to another organism and therefore change the observable characteristics of that organism.
4. Become familiar with sterile technique and decontamination procedures that are used to handle bacteria.
5. Learn how to calculate transformation efficiency.

Materials

Note: The pGREEN plasmid contains a mutant version of GFP linked to another gene called beta-galactosidase. This combination of genes was chosen because the protein produced from this combination turns bacteria yellow-green, even in normal light. If you expose the colonies to a UV light, they also fluoresce. The plasmid also contains the antibiotic resistance gene to allow growth in the presence of ampicillin.

The plasmid DNA should be kept in the refrigerator until it is aliquotted for students. When ready, have the students come up to you one at a time and dispense the plasmid DNA directly into the tubes they have labeled with a + to indicate that these tubes have the plasmid added.

Advance preparation

Day 1:

1. Follow directions on the E.coli vials to make plates 24 hours prior to lab.
2. Aliquot microtubes with just over 1 ml of 50 mM CaCl₂ to each be shared by two lab groups.
3. Make a 10% bleach solution to fill squirt bottles and the large disposal container.
4. Obtain enough crushed ice to fill foam cups or beakers for each lab group.
5. Fill water bath with distilled water and warm to 42°C.
6. Remove pGREEN plasmid DNA from freezer just before class begins.
7. Remove LB and LB/Amp plates from refrigerator and let warm to room temperature.
8. Pre-heat incubator to 37°C.
9. Aliquot one microtube with 1.5 ml of Luria broth for each two lab groups.
10. Have available enough squirt bottles with 10% bleach for every group to access.

Day 1 & 2:
Prepare a large container with 10% bleach solution for students to place their used pipettes and loops in and to sterilize their agar plates when transformation results have been analyzed.

Post Lab Cleanup

When all of your classes have finished using the streak petri plates, open the dishes and immerse in bleach solution for at least 20 minutes. Following sterilization, pour off liquid, put dishes in plastic bag, and throw away in regular trash.

Dispose of transformation petri dishes from day 2 in the same manner.

Teacher Notes

Information for Teacher

Use and disposal of E. coli:

Refer to the instructions which are provided with the E.coli vials.

E.coli is not considered pathogenic since it is a normal part of the bacterial flora of the human gut and is rarely associated with any illness in healthy individuals. As previously noted, the E. coli strain used in this experiment has been rendered non-pathogenic. However, it is very important to follow the guidelines below for handling of the bacteria and disposal of waste to ensure that the E.coli is used in a safe manner.

• Do not touch your face when handling plates or tubes with E. coli on them until you have washed your hands thoroughly. Do not put your face near the cultures or the tips when pipetting cultures.
  
  
• Do not incubate plates for more than the given time since this will allow contaminating bacteria and fungi to arise.
  
  
• Follow all directions for post-lab clean-up. Using gloves, collect all petri dishes, disposable pipets and tubes and immerse in a 10% bleach solution for 20 minutes or more to kill all bacteria. Drain excess solution, place materials in plastic bag and dispose in the regular
  garbage. DO NOT drip bleach on your clothes as it will permanently stain them.
  
  
• Wipe down lab bench with bleach solution at the end of lab.
  
  
• Wash hands thoroughly before leaving lab.

Student Activity: Transformation of the bacterium E. coli using a gene for green fluorescent protein

Background Reading

In molecular biology, transformation refers to a form of genetic exchange in which the genetic material carried by an individual cell is altered by incorporation of foreign (exogenous) DNA. This foreign DNA may be derived from unrelated species and even other kingdoms, such as bacteria, fungi, plants or animals, which would otherwise be inaccessible to an organism.

Bacteria and yeast have been transformed with human genes to produce proteins that are useful in treating human diseases and disorders e.g. the production of insulin. Some bacteria have been modified such that they are able to digest oil from accidental spills. Bacteria are single-celled organisms that can easily pass information between one another and thus changes in genetic make-up are rapidly passed on to subsequent generations.

Transformation is usually more difficult with multicellular organisms, such as plants, in which it is necessary to either alter many cells with the new piece of DNA or to alter just a single cell and then induce it to produce a whole new plant.

Genetic transformation of plants and other organisms does occur naturally. Bacteria and viruses can move DNA (or RNA) into an organism and cause profound changes. Examples are Agrobacterium tumefaciens (for plants) and HIV (for Humans).

The bacterium you will be transforming, E.coli, lives in the human gut and is a relatively simple and well understood organism. Its genetic material consists mostly of one large circle of DNA 4-5 million base pairs (mbp) in length, with small loops of DNA called plasmids, usually ranging from 5,000-10,000 base pairs in length, present in the cytoplasm. It is these plasmids that bacteria can transfer back and forth, allowing them to share genes among one another and thus to naturally adapt to new environments.

The ability of bacteria to maintain these plasmids and replicate them during normal cell multiplication is the basis of cell transformation. The plasmids are used as “gene taxis” in transformation events to bring DNA of interest into the cell where it can integrate into the genome or remain as a plasmid within a bacterium and be translated into proteins not normally found in that organism.

In this experiment, green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has been incorporated into a plasmid along with a gene for resistance to the antibiotic ampicillin. The GFP is actually located in discrete spots around the bell margin of the jellyfish and will fluoresce under certain conditions When inserted into a plasmid and used for the transformation procedure, the transformed bacteria will express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow green under ultraviolet light. The mutant form of GFP used in pGREEN makes the bacteria a yellow-green color even in white light.

This plasmid contains an ampicillin-resistance gene in addition to the GFP gene. Ampicillin is an antibiotic and works by preventing E.coli from constructing cell walls, thereby killing the bacteria. When the ampicillin-resistance gene is present, it directs the production of an enzyme that blocks the action of the ampicillin, and the bacteria are able to survive. Bacteria without the plasmid and, hence, the resistance gene are unable to grow on a plate containing ampicillin in the medium, and only the transformants will survive.

©Carolina Biological Supply Company, Used with permission.

GFP is also used in research as a reporter molecule. It can be linked to the protein that you are interested in studying, and this protein can then be followed through changes in expression of the linked GFP.

Objectives

1. Understand recombinant techniques and the transformation procedure using the heat shock method.
   
   
2. Understand how we can screen for a gene of interest and the importance of marker or reporter genes in molecular biology experiments.
   
   
3. Investigate how DNA can be transferred to another organism and the change in phenotype (physical characteristics) that may result from such a transfer.
   
   
4. Learn the importance of the sterile techniques that are used to handle bacteria, and the decontamination necessary when the experiment is complete.
   
   
5. Learn how to calculate transformation efficiency.

Materials

Precautions

Review the safety instructions with your teacher to ensure that you know how to handle the cultures and equipment safely. At the completion of the lab, dispose of all materials by soaking in 10% bleach solution and then draining and placing in the trash. Clean the lab benches with the bleach solution and remember to wash your hands before leaving the lab.

Procedure

Day 1:

1. Make sure that you have all of your group's materials, that you know which other group you will be sharing with, and where the bleach containers are located for clean-up at the end.
   
   
2. Put on gloves and safety goggles.
   
   
3. You have two sterile microtubes: mark one “+”and the other “-”. Write your lab group’s name or initials on each tube.
   
   
4. Using a disposable pipette, add 250 µl of 50mM CaCl₂ solution to each tube (“+” and “-”) and immediately place them both on ice.
   
   

   

   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
5. Use a sterile plastic loop to transfer one or two 3-mm bacterial colonies or an equivalent amount of smaller colonies from the streak plate to the “+” tube. Do not pick up any agar as it may inhibit the transformation process.
   
   
6. Immerse the loop tip into the calcium chloride solution in the “+” tube and vigorously spin the loop to dislodge the cell mass and disperse the entire mass into the calcium chloride solution. Exposure of the cells to cold calcium chloride solution, in combination with the "heat shock" discussed in step 12 below, causes the cell membrane to become porous and thus the cells are made "competent" for transformation. Note: No visible clumps of cells should remain. This step is critical to obtaining good results.
   
   
7. Place the loop into the bacterial waste container to kill the bacteria that remain on it.
   
   
8. Close the tube lid and put the tube on ice.
   
   
9. Follow steps 5 to 8 and use a new sterile loop to transfer a mass of cells to the“-” tube. Both tubes should now be on ice.
   
   
10. Using a sterile inoculating loop pick up one loopful (10 µl) of pGREEN and add directly into the CaCl₂ in your “+” tube. Your teacher may do this for you. Following the addition of the plasmid, close the tube lid and tap gently with your finger to mix. Try not to splash the suspension up the sides of the tube. Tap the base of the tube gently on the desktop to make all of the liquid move to the bottom of the tube.
    
    
11. Return the “+” tube to the ice. DO NOT add the plasmid to your "-" tube. Incubate both tubes on ice for 15 minutes.
    
    
12. While the cells are incubating, your teacher will pass a UV lamp over the pGREEN DNA solution. Note your observations on the student activity sheet and complete questions 1-3.
    
    
13. Following incubation, "heat shock" the cells. It is essential that the cells receive a sharp and distinct shock.
    a. Carry the ice container with the tubes to the 42°C water bath.
    b. Remove both tubes from ice and immediately hold them in the water for 90 seconds. Three-fourths of the tube should be under water.
    c. After heat shock, immediately return the tubes to ice. Let them stay on ice for at least one minute.

    Stop Point: The tubes can be refrigerated overnight and removed just prior to the beginning of the next lab. If you will not finish the lab today, give the tubes to your teacher for overnight storage. Clean up: Place used loops etc in the bacterial waste container. Spray workspace with bleach solution and wipe with paper towels. Wash hands before leaving lab.

14. Use a disposable pipette to add 250 µl of Luria broth to both the "+" and "-" tubes. Make sure that you add to the "-" tube first so as to avoid cross-contamination of the plasmid. Discard this pipette into the bacterial waste.
    
    
15. Close lids, and gently tap tubes to mix. Place in a rack and incubate for 10 minutes at room temperature.
    
    
16. Label the BOTTOM of your media plates while the tubes are incubating. Make sure to also include your lab group name and the date.
    
    
17. Label 1 LB plate and 1 LB/Amp plate “+PLASMID” and label 1 LB plate and 1 LB/Amp plate “-PLASMID”
    
    
18. Use a disposable pipette to add 100 µl of cell suspension from the “- tube” to the “LB - PLASMID” plate, and another 100 µl to the “LB/Amp - PLASMID” plate. Discard the pipette and “- tube” into the bacterial waste container.
    
    
19. Using a new sterile loop for each plate, spread the suspensions evenly around the surface of the agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface. Turn the plate a quarter turn and go back and forth several more times.
    
    
20. Use a new sterile pipette to transfer 100 µl of cell suspension from the "+" tube to each appropriate plate and spread as above.
    
    
    
    
    
    
21. Rest the plates on the bench for 10 minutes to allow the cell suspension to absorb into the medium.
    
    
22. Wrap Parafilm around your four plates to seal the lids. Place the plates upside down in an incubator or at room temperature. The results will be ready to observe after 24 hours if incubated at 37°C or after 48-72 hours if incubated at room temperature.
    
    
23. After tips and tubes have sat in bleach solution for at least 20 minutes, pour liquid in bacterial waste container down the drain. Collect tips and tubes in a plastic bag and discard in the normal garbage. Spray down workspace with bleach solution. Wash hands before leaving lab.

Day 2:

1. Retrieve your plates from incubator or other storage area and check for growth. Complete the remainder of the activity sheet you began on day 1 of the lab.
   
   
2. Open the petri plates and immerse in the large container of bleach solution your instructor has provided. Spray down workspace with bleach solution. Wash hands before leaving lab.

Student Activity

1. What color was the pGREEN plasmid DNA when we exposed it to the UV light?
   
   
2. Will all of the plates have bacteria growing on them?
   
   
3. Explain your answer to question 2.
   
   
4. Now observe the results on the petri dishes without removing the lids. Count how many colonies are present on each plate. If there are too many to count then record "lawn". Record your results on the diagrams below.
   
   
   

   

   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
5. Were your results different from what you expected? Explain what you think might have happened.
   
   
6. Which plates are the control plates? Why do we need the control plates?
   
   
7. Only one plate has transformants. This is the experimental plate. Which one is it? Why do only transformants grow on this plate?
   
   
8. Shine the UV light on the plate with the transformants. What color are the bacteria? Compare this answer with what you observed when the light was shined onto the pGREEN plasmid. Is there a difference? Why?
   
   
9. Were all of the bacteria that we started with transformed? Compare the growth on plates 2 and 4 and then explain your answer.
   
   
10. Transformation efficiency is defined as the number of transformed colonies per microgram (µg) of plasmid DNA. In order to determine the efficiency of the transformation we need to determine the initial amount (mass) of plasmid that was spread on the plate and relate this to the number of transformed colonies that were observed on the experimental plate.
    
    The formula for determining the transformation efficiency is:
    
    Total number of colonies growing on the LB/amp per µg of plasmid DNA used for the transformation
    
    a) To determine the initial amount (mass) of plasmid DNA:
    Total amount of DNA [µg] = concentration [µg/µl] X volume [µl] Remember, you used 10 µl of plasmid DNA at a concentration of 0.005 µg/µl
    
    
    b) To determine fraction of DNA solution spread:
    Volume of solution spread on plate divided by total amount in tube = fraction spread
    
    
    c) To determine the amount of DNA spread on the experimental plate:
    Total amount of plasmid DNA [µg] X fraction spread = amount of DNA spread
    
    
    d) Number of colonies per µg of plasmid DNA:
    Number of colonies observed divided by amount of DNA spread = Transformation efficiency

Answers to Student Activity

1. The plasmid does not fluoresce green.
   
   
2. No
   
   
3. The bacterium cannot grow in the presence of the antibiotic ampicillin unless it contains the plasmid, and so there will be no growth on the LB/Amp plate of the bacteria without the plasmid.
   
   
4. See image above.
   
   
5. On plate 1 since there is no antibiotic present the wild-type bacteria grew normally and formed a "lawn" across the whole plate.
   
   
   On plate 2 the transformed bacteria can still grow to form a "lawn" since there is no antibiotic present to inhibit growth of bacteria without the plasmid.
   On plate 3 the untransformed bacteria are unable to grow in the presence of the antibiotic since bacterial cell walls are unable to form and the bacterial cells die.
   On plate 4 any bacteria that has been transformed by taking up the pGREEN plasmid is now able to grow in the presence of the antibiotic since the plasmid also contains the gene allowing for antibiotic resistance. These bacteria will also fluoresce green.
   
   
6. Plates number 1 and 2 are positive controls while plate number 3 is the negative control. Each of these plates tests a different combination of components of the experiment to verify that they are all functioning as expected.
   
   
7. Plate number 4 has the transformants since this is the plate that contains the antibiotic that allows only bacteria containing the pGREEN plasmid and its antibiotic resistance gene to grow.
   
   
8. The transformed bacterial colonies glow green when illuminated with the UV light. There was no fluorescence visible when the UV light was shined on the plasmid in the tube because it is not the plasmid that reacts to the light but the protein produced when the gene in the plasmid is translated. This protein production only occurs once the plasmid has been incorporated into the bacteria.
   
   
9. If all of the bacteria were transformed then plate 4 would have a "lawn" of growth, the same as plate 2. Only a limited number of the bacteria actually incorporate the plasmid into their cells during the course of this experiment.
   
   
10. a) Total mass = 10 µl x 0.005 µg/µl = 0.05 µg
    b) Fraction spread = 100 µl / 510 µl = 0.196
    c) Mass of plasmid DNA spread = 0.05 µg x 0.196 = 0.0098 µg
    d) Transformation efficiency = x colonies on plate 4/0.0098.
    The answers will vary but should be in the range of 10³ -10⁴ transformants/µg DNA.

Note: Factors influencing transformation efficiency include technique errors, the temperature and length of the incubation period, the growth stage of the cells, and using the correct mass of plasmid DNA.

The protocol above has been modified from UC Davis. The website below provides information and pictures of the transformation and regeneration of a tomato explant using Agrobacterium tumefaciens to carry the engineered DNA into the tomato cell. http://ppge.ucdavis.edu