What is genetic engineering? How to make glow-in-the-dark bacteria (Traditional method)- Teacher Guide

Photos by Faith Carter

What is genetic engineering, and how is this technique used? Students perform a genetic engineering experiment using bacterial transformation to intorduce a bioluminescence gene into E. coli. Students transform E. coli with a plasmid that contains the lux bioluminescence gene to produce bacteria that glow in the dark. This activity helps students understand what genes do and how they can be manipulated by genetic engineering.

Classroom time needed for this lab:

• two class periods (~50 minutes each)

Setting up the classroom:

Distribute for each pair of students:

• 2 microcentrifuge tubes (1.5 ml size) of competent E. coli cells in calcium chloride solution (100 ul per tube)
• 1 microcentrifuge tube (0.5 or 0.65 ml size) containing 5 ul pUC18 DNA (10 ng/ul)
• 1 microcentrifuge tube (0.5 or 0.65 ml size) containing 5 ul pLux DNA (10 ng/ul)
• 3 ml LB broth in sterile tube
• 2 LB agar + ampicillin plates
• Graduate micropipets with tips
• 2 sterile 1 ml disposable transfer pipets
• 2 sterile 0.3 ml disposable transfer pipets
• Container of ice
• Permanent marker
• Sterile cotton swabs

Have a 37⁰C incubator (for growing bacteria) and 37⁰C water bath ready (for heat shock). If a water bath is not available, an incubator could be used for the heat shock step, but this is less accurate. You will need large waste beakers at the lab stations for disposing tips, transfer pipets, and cotton swabs. A biological waste bag is also needed for disposal of bacterial plates.

You will need to prepare the following materials (recipes to follow):

Ampicillin (100 mg/ml)
LB agar + ampicillin plates
LB broth
Sterile toothpicks and flasks
Sterile 50 mM calcium chloride

During the lab:

Make sure that all materials contaminated with bacteria are disposed properly. After the plates are incubated overnight, you can store the plates containing bacteria at room temperature for a day or so until the class meets again. If any more time required, store the bacterial plates in the refrigerator.

Preparing LB agar plates with ampicillin

Ampicillin (100 mg/ml solution)

1. Dissolve 5 g of ampicillin (sodium salt, MW = 371.40) in 50 ml of deionized or distilled water.
   
   
2. Prewash a 0.45- or 0.22-micron sterile filter by drawing through filter 50 ml of deionized or distilled water. Pass the ampicillin solution through the filter and dispense 10-ml aliquots in sterile capped tubes or bottles. Freeze at -20⁰C.
   
   

LB agar plates with ampicillin

1. Add 32 g of LB agar mix (Life Technologies) to two liter flask:
   
   Note: If you do not have a two liter flask, you can use 500 ml or 1000 ml flasks. Be sure to adjust the amounts proportionally (i.e., 500 ml of media in 1000 ml flask, 250 ml of media in 500 ml flask).
   
   
2. Add 1 liter of deionized or distilled water and mix to dissolve ingredients, preferably using a magnetic stirrer. The agar in the solution will not dissolve.
   
   
3. Cover the top of the flask with aluminum foil, and autoclave the solution for 20 minutes at 121⁰C. If you use a pressure cooker, you need to sterilize at 15 lbs pressure for 20 minutes, and temperature should reach 121⁰C or 250⁰F.
   
   
4. After autoclaving, swirl LB agar solution to distribute agar evenly.
   
   
5. Let the solution cool just until the flask can be held in your hands (55-60⁰C). If the solution cools too long and begins to solidy, remelt it by briefly autoclaving for 5 minutes or less.
   
   
6. Add 1 ml of the 100 mg/ml ampicillin to the 1 liter of LB agar solution. Antibiotics are sensitive to heat, so it is important to cool the solution to 55-60⁰C before adding the antibiotic.
   
   
7. Swirl the flask to mix the ampicillin.
   
   
8. Mark sterile culture plates with description of media. If you are using presterilized polystyrene plates, the the plastic bag can be used for storing the poured plates.
   
   
9. To pour media into plates, carefully the lift the lid of the culture plate to pour the solution. Do not set the lid on the lab bench. Quickly pour in agar until the bottom plate is about 1/3 full. Immediately replace the lid. Repeat procedure for each plate, and occasionally flame the top of the flask. Always hold the flask slightly tilted to prevent airborne contaminants from falling into the flask.
   
   
10. Let the agar solidify, and store the plates lidside down overnight to dry the agar and minimize condensation.
    
    
11. Store the plates in a covered container or in the plastic bags in the refrigerator. The ampicillin in the media is effective for 1-2 months when stored in the refrigerator.
    
    

Luria-Bertani (LB) broth

1. Add 20 g of LB broth mix (Life Technologies) to a 2 liter flask.
   
   

   Note: If you do not have a two liter flask, you can use 500 ml or 1000 ml flasks. Be sure to adjust the amounts of reagents proportionally (i.e., 500 ml of media in 1000 ml flask, 250 ml of media in 500 ml flask).

   
   
   
2. Add 1 liter of deionized or distilled water and mix to dissolve ingredients, preferably using a magnetic stirrer.
   
   
3. Divide the LB broth among several bottles (for example, 200 ml LB in 250 ml bottles or 100 ml LB in 125 ml bottles). Put on the caps loosely.
   
   
4. Autoclave the solution for 20 minutes at 121⁰C. If you use a pressure cooker, you need to sterilize at 15 lbs pressure for 20 minutes, and temperature should reach 121⁰C or 250⁰F.
   
   
5. Let the solution cool and tighten the caps. The LB broth can be stored at room temperature indefinitely.
   
   

Note: If the broth becomes cloudy after storage, this is a sign of microbial contamination, and the broth needs to be disposed. Add bleach to the broth (to a final concentration of ~10%), let stand for 15 minutes or more, then dispose in the sink.

Preparing competent E. coli cells

This is technique requires special laboratory equipment and involves several steps. The cells, however, have a much higher transformation efficiency than the rapid colony method.

50 mM calcium chloride

1. Dissolve 5.6 g of anhydrous CaCl2 (m.w. 110.99) or 7.4 g of the dihydrate form (m.w. 146.99) in 180 ml deionized or distilled water.
   
   
2. Add deionized or distilled water to make 200 ml of total solution. This solution, at 500 mM concentration, can be stored in a capped bottle in the refrigerator.
   
   
3. Mix 50 ml of the 500 mM calcium chloride solution with 450 ml deionized or distilled water. The concentration of calcium chloride is now 50 mM.
   
   
4. Dispense the CaCl2 solution (50 mM) into capped glass bottles. Autoclave or use a pressure cooker (20 minutes at 121⁰C at 15 psi.) with the bottles loosely capped.
   
   
5. After the bottles cool, tighten the bottle caps and store in the refrigerator. The solution will be cooled and ready for making competent cells (see below).
   
   

Sterile toothpicks, cotton swabs, and flasks

1. Use flat toothpicks (not the pointed ones). Place flat toothpicks in a 50 ml glass beaker, with the wider end of the toothpicks at the bottom of a 50 ml beaker. Cover the beaker with aluminum foil.
   
   
2. Use cotton swabs with a wooden stick. Put cotton swabs in glass beaker, with cotton end at the bottom of beaker. Cover beaker with aluminum foil.
   
   
3. Put a magnetic stir bar into an empty 125 ml flask. Cover mouth of flask with foil.
   
   
4. The toothpicks, cotton swabs, and flasks can be sterilized by two methods: a) Autoclave the beaker for 20 minutes at 121⁰C at 15 psi or b) Bake in a oven at 300⁰F for at least an hour.
   
   

Plating bacteria for single colonies

1. Use a permanent marker to label the bottom of an LB agar plate with the date and strain used.
   
   
2. Pick a sterile toothpick from the container. The wider end of the toothpick is used for bacteria; the narrower end is held in your fingers. Remove the lid from the E. coli culture plate with your free hand. Do not place the lid on the lab bench. Hold the lid face down a little bit above the culture plate to prevent contaminants from falling onto the plate. Gently scrape a small mass of bacteria onto the toothpick.
   
   
3. In a small area of the plate, carefully spread the bacteria onto the agar surface. Try not to gouge the agar surface (it helps to hold the toothpick nearly parallel to the plate surface when spreading bacteria). After spreading bacteria, dispose toothpick in a waste container.
   
   
4. Using a fresh sterile toothpick, glide the end of the toothpick two times across the first bacterial streak, and then spread the bacteria on the toothpick on an adjacent section of the plate using a tight zigzag streak. This streak should cover about a third of the agar surface.
   
   
5. Using a fresh sterile toothpick, glide the end of the toothpick two times across the last bacterial streak, and then spread the bacteria on the toothpick on an adjacent third of the agar surface using a tight zigzag streak.
   
   
6. Incubate the plates upside down in a 37⁰C incubator overnight. You should see single colonies on the last series of streaks across the plate.
   
   

Growing E. coli in liquid cultures

1. Gently scrape one bacterial colony off the LB agar plate using a sterile toothpick.
   
   
2. Stir the toothpick into 4- 5 ml LB broth in a sterile test tube with cap.
   
   
3. Incubate test tube in a test tube shaker at 37⁰C overnight.
   
   
4. After incubation, the broth should be cloudy with bacterial growth.
   
   

Note: If you do not have access to a test tube shaker, you can use a magnet stirrer and incubator. Inoculate a single colony into a sterile, foil-covered 125 ml flask containing 25 ml sterile LB broth and magnetic stir bar at bottom. Put the stirrer and flask into a 37⁰C incubator and stir overnight. The broth should be cloudy with bacterial growth the next day.

Preparation of competent E. coli cells

1. Dilute 0.5 ml of an overnight culture of E. coli into 25 ml LB broth in a foil-covered sterile 125 ml flask. Incubate cultures on shaker at 37⁰C with mixing until cells are at mid-log stage (takes about 2.5 hours). You can use a magnetic stirrer and incubator (see above).
   
   
2. Add 10 ml of mid-log culture into 15 ml capped tube. This will produce enough cells for about 10 transformation reactions. Centrifuge tubes at 500-1000 x g for 10 minutes (these speeds can be obtained with a clinical centrifuge). Cells should form a pellet at the bottom of tube. If cells are not pelleted, then respin tubes.
   
   
3. Carefully pour off the supernatant into a waste beaker. Invert the tube onto a paper towel to allow the supernatant to drain.
   
   
4. Add 5 ml of ice-cold calcium chloride to the tube. Pipet the solution several times to resuspend the cell pellet. The cells should be resuspended completely.
   
   
5. Keep the tube on ice for 20 minutes.
   
   
6. Respin the cells in a centrifuge for 5 minutes at 500-1000 x g.
   
   
7. Carefully pour off the supernatant into a waste beaker. Be sure not to disturb the cell pellet which may be fragile at this step. Invert the tube onto a paper towel to allow the supernatant to drain.
   
   
8. Add 1 ml of ice-cold calcium chloride solution to the tube. Resuspend the cells in the solution by pipeting or several times. No cell clumps should be present in the mixture. Divide the cells into 100 ul aliquots in 1.5 ml size microcentrifuge tubes. The tubes can be stored on ice in the refrigerator for up to a week (DON'T FREEZE THE CELLS).
   
   
9. Clean up contaminated materials by soaking them in 10% bleach for 15 minutes or more before washing.
   
   

Sources of laboratory materials