Transcription and Gene Expression in Bacteria
Student Laboratory Kit
Materials Included In Kit
Bleach solution, sodium hypochlorite, NaOCl, 500 mL
Nutrient agar, 25 g
Petri dishes, plastic, sterile, 30
Sterile cotton swabs, 60
Additional Materials Required
(for each lab group)
Water, spring or tap, 600 mL
Water, sterile, 100 mL
Autoclave or pressure cooker
Erlenmeyer flask, 1000-mL
Heat resistant gloves (for preparation of agar)
Hot water bath or microwave
Plug, foam, 45–65 mm
Serratia marcescens culture, 1 tube
Spray bottle (for cleanup)
Making and pouring the nutrient agar should be done at least one day before the lab. Add only enough nutrient agar to cover the bottom of each Petri dish (about 20 mL).
Preparation of nutrient agar: Mix 15 g of nutrient agar with 600 mL of water in an Erlenmeyer flask. Place a foam plug in the mouth of the Erlenmeyer flask to prevent evaporation of the water. Bring the solution to a boil, stirring occasionally to completely dissolve the agar. Sterilize the nutrient agar using an autoclave or pressure cooker. Sterilize according to the instruction manual or at 121 °C at 15 psi for 15 minutes.
To help prevent contamination before and after pouring the plates, follow these steps:
- Disinfect work areas using a spray bottle containing a bleach solution or liquid Lysol®.
- Open the sealed sleeve of plastic, sterile, Petri dishes once the nutrient agar is read to be poured. (You may wish to keep this sleeve for storing the plates after they have been poured and cooled.)
- Without removing lids, put the dishes in a single line on the outer edge of the work area.
- Using insulated gloves, pour approximately 15 mL of nutrient agar into each Petri dish by picking the lid straight up, keeping the lid between you and the liquid agar. Hold your breath while pouring each dish to help prevent airborne contamination. Add only enough nutrient agar to cover the bottom of the dish. Replace the lid. Avoid excessive movement to prevent creating air drafts.
- Allow the nutrient agar to cool without disturbing the dishes in any way. (Depending on room temperature, this could take up to 30 minutes.) When the dishes are completely cooled, tape them shut with two pieces of tape. Turn the dishes upside down and put them back in the plastic sleeve. Tape the sleeve shut and put the entire sleeve in a refrigerator or other cool location. Do not freeze.
- Prior to handing out the dishes to students, check for contamination on the nutrient agar surface. If contamination is present, tape the dish(es) shut and dispose of accordingly. Set the incubator to 37 °C a couple of hours before beginning the activity.
Although Serratia marcescens is a non-pathogenic bacterium, there is always the potential for contamination by pathogens when dealing with microorganisms. Instruct students to wear goggles and gloves and to follow aseptic techniques when handling microbes. Do not allow students to open the Petri dish lids after inoculation. Remind students to disinfect the work surfaces and to wash their hands thoroughly with antibacterial soap and water after working with microorganisms. Sodium hypochlorite provided in the kit for disinfection is corrosive and toxic. Avoid contact with acids, which can release toxic chlorine gas.
Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Upon finishing work with bacterial cultures, label the stock tubes and instruct students to disinfect their work areas (including the incubator handle). Use a spray bottle containing the bleach solution included with the kit (if the surfaces are compatible with bleach), or a 70% isopropyl alcohol solution or Lysol® solution to sterilize all work surfaces. Microbiological cultures may be disposed of according to Flinn Suggested Biological Waste Disposal Method Type I.
- Enough materials are provided in this kit for 30 students working in pairs, or for 15 groups of students. Bacterial colonies should be visible after 48–72 hours. The Prelab Questions should be completed before beginning the activity, and students should be allowed an extra day after making observation to answer the Post-Lab Questions.
- Tyndallization can be used if no autoclave is available. Boil the nutrient agar for 20 minutes in the covered Erlenmeyer flask. Cool the agar and incubate at 37 °C for a day. Repeat the boiling, cooling, incubation procedure for three consecutive days followed by boiling again. The three incubations force many of the heat-resistant spores to grow due to heat shock. These newly growing bacteria and fungi are killed in the next boiling step.
- The “25 °C” dishes may be stored at room temperature, which is generally in the range of 20–25 °C. If preferred, the exact temperature of the room may be measured and written on the dish rather than 25 °C. Optimal color is observed in the range of 24–28 °C.
- Cultures grown at room temperature should have smooth, dark red bacterial colonies present. Cultures grown at 37 °C should display white or pale pink colonies. To achieve all white colonies, subculture the 37 °C Petri dish onto a fresh Petri dish and incubate at 37 °C for 48 hours.
- Stock cultures of S. marcescens contain red-colored colonies. However, if the bacteria are shipped in hot weather they may be pink or white upon arrival. If you do not want students to see the original color of the bacteria, place black electrical tape around the tube and dip the sampling swabs into the tube for the students.
- As an alternative or follow-up procedure, students may experiment with growing S. marcescens cultures at different temperatures between room temperature and body temperature to determine the temperature cutoff for the expression of the red pigment. Cultures grown at temperatures in between those tested in this activity are often pink in color. This is because some cells are still expressing the prodigiosin but many are not.
Answers to Prelab Questions
- Why is it considered proper practice to label the bottom of the Petri dish rather than the lid?
Lids may be accidentally interchanged between Petri dishes. The bottom of the dish cannot “move” and therefore will identify the bacteria sample and “owner” in the event that the lid is misplaced, cracked, etc.
- Explain the term gene expression in your own words.
Gene expression refers to the transcription of gene DNA to produce mRNA, which is ultimately translated into the proteins required for specific cellular processes. The genotype encoded in the gene is “expressed” in the phenotype.
- Write a hypothesis for what you predict will be observed at the two different temperatures based on information in the Background section.
The culture grown at 25 °C may contain the prodigiosin pigment and appear red in color. Cultures grown at 37 °C may lack the enzyme precursors that form prodigiosin and therefore appear white or colorless. This hypothesis is based on the sentence in the Background section that states, “Studies indicate that the prodigiosin protein may have anti-fungal properties, which may help the bacteria ward off fungal “competitors” that utilize common resources in the soil.” Soil is likely to be a much cooler environment than the human body.
Answers to Questions
- Draw what was observed on each of the two plates. Use colored pencils, or describe in words, the colors observed.
- Based on the observed results of this activity, predict what the result would be if a culture of Serratia marcescens were grown at a temperature between the two temperatures tested in this activity. Explain your reasoning.
In the Background section, it states that the bacteria may appear red, pink, white or colorless. In a temperature range midway between 25 °C and 37 °C, the bacteria are likely to appear pink as the enzymes responsible for the synthesis of prodigiosin are not being transcribed at a lesser rate.
- If someone had a population of Serratia marcescens present on their skin and they came into contact with rhizopus mold, would the presence of S. marcescens offer resistance to the mold? Explain.
If the bacteria were present on the hand, then they would be thriving at 32–33 °C (average surface temperature of the body) and prodigiosin would likely not be produced by the bacteria, or be produced at low rates. Since it is specifically the prodigiosin protein that may have anti-fungal properties, not the S. marcescens bacteria in general, it is not likely that any anti-fungal properties will be in effect.
- Suggest a possible advantage for the S. marcescens to have a temperature-sensitive protein like prodigiosin? In your opinion, why would a temperature dependant pigment be and evolutionary advantage?
For energy and resource conservation—so that energy for the production of the prodigiosin protein is conserved for times when the bacteria may have greater competition for resources.
- Based on the incubation temperature in which prodigiosin was produced, what do you believe the optimal growth temperature is for competitors of S. marcescens?
Around 25 °C