Lights On - We Have A Quorum

Roger S. Schoob

Overview

This series of bacteriological laboratory experiences will acquaint students with basic bacteriological techniques. The student’s observations should also help them to be able to visualize communication on a simple cell to cell level. The students should become aware of the environmental and evolutionary implications of this communication. There are many similarities to intercellular communications in multicellular eukaryotic organisms. The extensions can culminate in investigating the genetic level of this communication by genetic manipulation of the organism. If you have the necessary equipment and expertise, restriction analysis and genetic transformation can be a logical extension activity.

Biological Concepts

• Aseptic technique

• Colony cloning

• Serial dilutions

• Bacterial communications

• Bacterial conjugation

• Bacterial transformation

• Environmental influence on metabolism ( extension )

Class Time

If the students are well prepped (see student lab sheets) the streaking and serial dilutions can be accomplished in less than one laboratory period each. Additional time is needed for observation and discussion. The bacterial conjugation and bacterial signalling labs will also take a period (or less) to set up and additional time to observe and discuss. If you have the equipment for the DNA isolation and restriction digests and recombination of competent (transformable E. coli) cells, this bacterial system will work well for you. These higher level labs take substantially longer and you need some experience in determining suitable stopping points for your lab schedule.

Background Information (luxO) allowing an activator (luxR) to promote transcription of the luminescence gene (luxCDABEGH) .

STREAK PLATES - V. harveyi is easy to culture and mutants are available which allow for the investigation of its communication abilities. The first activity should be to do two streak plates. (See student lab sheets.) One to isolate individual colonies by standard three or four overlapping streaks with sterilization between each streak. The second should be done with sterile cotton swabs, allowing the students to draw names, initials, smiles or any nonobjectionable design on the plate with the culture. The glowing results the next day will have them hooked for the follow-up discussions.

SERIAL DILUTIONS - The bacterial colonies are glowing after overnight incubation. Younger colonies will not glow.(You might want to have a 12 hour culture for comparison) Why? It takes a certain cell density (a quorum) to signal luminescence. The number necessary to reach a quorum is ~ 10 ⁹ . It is probably important to describe the difference between luminescence and fluorescence at this time. These luminescent bacteria produce their own light from an energy consuming metabolic pathway. fluorescence occurs when certain chemicals emit light of a different wavelength after being exposed to some light source. How many are in one colony? How many does it take to glow? How can we count them? It is time to introduce serial dilutions. ( See student lab sheets) >where it can be expressed. This transfer happens with a fairly low frequency, but the numbers involved are so high that a large number of conjugates will result. To find these cells we will also introduce the concepts of selecting and screening the potential conjugates to determine those that have undergone the desired genetic recombination. The V. harveyi is naturally ampicillin resistant. The transferred plasmid contained an inserted gene for tetracycline resistance. All of the E. coli should be susceptible to ampicillin and die on ampicillin + tetracycline plates. Unchanged V. harveyi in the experimental group will not tolerate tetracycline and die. Only V. harveyi that have taken up the plasmid will survive this selection process and should then also express the new luminescence genes after incubation. If they do luminesce then they are expressing a transferred gene and we can then use that expression to screen for those colonies that originated from the successful conjugates. Successful conjugates in the control group will also grow, but they will not glow since their plasmid does not contain the luminescence genes. Topics of discussion to follow this lab might include the natural intra and inter species transfer of antibiotic resistance; gene therapy to correct errors in metabolic pathways; the ability to use bacteria as models for more complex systems and the ability to transform a bacteria to make something it was not making (often a useful product). This last concept can be even better illustrated by a traditional transformation - of E. coli to produce light. This is more complex, but the information necessary for those familiar with the process will follow.

TRANSFORMATION - For those comfortable with the biotechnology procedures and having the necessary equipment, competent E. coli can be transformed to become luminescent with plasmid pJE202 available from Life Technologies. The resulting transformed cells can be lysed and the plasmids digested with HindIII . This digest can be compared to stock pJE202 and pBR322. This series of laboratory experiments will give those students doing advanced high school biotechnology a real feel for the connectedness of the whole process of bacterial transformations. Again this can be tied to discussions of selection, screening and the importance of bacterial transformations in producing products not indigenous to the organism, such as bacterially produced insulin, hemoglobin, proteinases, and enzymes for bioremediation, food production and other industrial uses.

CROSS "FEEDING" - A final easy demonstration of the nature of the cell to cell communication involved in this system involves two V. harveyi mutants. One strain, BB151, is not luminescent because it lacks the gene complex for the luminescence substrate and enzymes, it does have the genes for the autoinducers, or exogenous signal molecules. The other strain, BB202B, has the luminescence gene complex, but lacks the signalling ability, because it has defective autoinducer genes. Neither strain can produce light when grown alone. When placed near each other the autoinducer from the donor,BB151, signals the recipient, BB202B, that there is a large number of V. harveyi present and BB202B lights up. (See student lab sheet) When left out overnight in the lab there should be a line of luminescence by morning. Over time the luminescence will spread further into the BB202B culture, but will dim as resources are depleted.

Materials:

Antibiotic Heart Infusion Agar (for selection of V. harveyi)

25 g Heart Infusion Medium

15 g Sodium Chloride

20 g Bacto Agar

1000 ml water

Autoclave 20-30 min and cool to ~55^oC.(the antibiotics are heat sensitive)

Add 1 ml of 100mg/ml Ampicillin (dissolved in water) and 1 ml of 10 mg/ml Tetracycline (dissolved in methyl alcohol)

Swirl the media and pour into sterile petri plates. Makes ~ 40 plates.

(To make HI broth omit agar and antibiotics )

Luria Broth (for E. coli culture and making LB agar plates)

10 g Tryptone

5 g Yeast Extract

10 g Sodium Chloride

1000 ml water

Autoclave 20-30 min.

(15 g agar if making plates)

Add antibiotics from above if using for E. coli transformations

Bacterial Cultures

Vibrio harveyi

BB721 (Lux⁺ for serial dilutions and bio-assays) ATCC

BB151 (Lux^- for recipient in conjugation) ATCC

BB202B ( Autoinducer ^- for recipient in intercellular signalling ) ATCC

Escherichia coli

BB1 (pLAFR2 with luxCDABE donor in conjugation ) ATCC

BB2 ( helper in conjugation, produces sex pilus) ATCC

BB3 ( control in conjugation, pLAFR2 without luxCDABE) ATCC

BB4 (pBR322 for use in DNA isolation - control for JE202) ATCC

JE202 (pBR322 with the Vibrio luminescence genes cloned into it) Life Technologies

Plasmids

pBR322 (common plasmid used in cloning) ATCC

pJE202 (pBR322 with Vibrio luminescence genes cloned into it) Life Technologies

Extensions/Variations:

After seeing the capabilities of V. harveyi there are many ways to modify the preceding experiments. Since the luminescence is a metabolic pathway it would be interesting to determine the effects of a variety of chemicals and conditions on that pathway by relating changes to resultant changes in light production. Factors that might affect these pathways or signalling abilities include: iron, carbon source and concentration, sodium chloride, calcium, caffeine, nicotine, neurotransmitters, heavy metal compounds or water that might be chemically polluted. Population studies might determine the time that the culture reaches the critical density for light production. Differences in light quality or intensity might be measured.

Resources:

American Type Culture Collection (A source for bacterial cultures at the cost for shipping)

12301 Park Lawn Drive

Rockville, MD 20852-1776

fax (301) 816-4365

Bassler, Bonnie and Silverman, Michael R. "Intercellular Communication in Marine Vibrio Species: Density-Dependent Regulation of the Expression of Bioluminescence, Two Component Signal Transduction, James A. Hoch and Thomas J. Silhavy, Ed. American Society for Microbiology , Washington, D.C. 1995 ( An in depth review of this system)

Life Technologies ( Source for plasmids not available from ATCC)

Gaithersberg, MD

(302) 840-8000

(800) 828-6886 ( tech support and ordering)

Miklos, David A. and Freyer, Greg A., DNA Science: A First Course in Recombinant DNA Technology, Cold Springs Harbor Laboratory Press, Cold Springs Harbor, N.Y. and Carolina Biological Supply Co., Burlington, N.C., 1990.

Acknowledgments:

I would like to thank Bonnie Bassler Ph.D. of Princeton University for her ideas, time, enthusiasm and cultures.

About The Author:

Roger Schoob is a biology teacher at Bolingbrook High School in Bolingbrook, Illinois. Roger can be contacted at Bolingbrook High School, 305 Blair Lane, Bolingbrook, IL 60440 or by e-mail at rschoob@aol.com.

Bacterial Streaking and Painting

Overview: In order to work with bacteria we need to know that we have what we want where we want it. We take precautions to be sure that we only put what we want into a sterile environment. In order to be sure we are starting with one type of bacteria we will start our cultures from a single colony of bacteria. Each colony represents the clonal offspring of one bacterium. If the colonies are well separated we can pick them off and start pure cultures. To obtain individual colonies we will spread out a culture on an agar surface. This is often done by a technique called streaking.

• Use a sterile loop (flamed or disposable) to transfer bacteria from a stock culture of Vibrio

harveyi BB721, to a streak on a sterile petri plate of HI (heart infusion) agar. Lift the lid

as little as possible and make two to three passes on one third of the plate. (see figure 1)

• Use a sterile loop to cross the first streaks with two or three new streaks. Do not get more stock culture onto your loop, you are now just spreading the first streak out. (see figure 2)

• Use a sterile loop to cross the second streak twice and spread onto the open agar without

crossing any of the first streaks (see figure 3)

• HINTS: Streaks are hard to see. It is easier to tell where you have streaked by turning the plate to always reach in the same way. (see figures 1-3)

PAINT: Use a sterile cotton swab to transfer V. harveyi BB721 to a sterile HI agar plate. Draw something you wish to see with the swab. It must be something you KNOW your teacher is not going to object to.

What do you see on your streak plate? Why?

Sketch your results.

Did you have any problems? What were they and how could you correct them?

Serial Dilutions

USING ASEPTIC TECHNIQUES:

• Prepare 10 culture tubes containing 4.5ml of HI liquid medium.

• Add 500ul of the overnight culture of V. harveyi to the first tube and mix (1:10 dilution).

A sterile pipette tip is required for each transfer. If micropippets are not available sterile

Pasteur pipettes or disposable plastic can be calibrated to deliver ~500ul.

• Transfer 500ul of the 1:10 dilution to the next tube to yield a 1:100 dilution and mix.

• Repeat for all ten tubes. Be certain to change tips. You now have dilutions from 10¹ to

10¹⁰.

• Spread 100ul of each dilution onto 10 HI agar plates. Sterilize a glass spreader in alcohol and flame in a Bunsen burner to remove the alcohol. Cool the spreader on the agar (away from the bacteria) and spread the bacteria over the surface. Working in groups, each student should get to do several plates.

• Let the liquid soak into the agar. Invert the plates and incubate overnight at 30^oC. (Or on

the bench top)

• Count the colonies the next day to determine the original concentration. The number of

bacteria per ml equals the number of colonies counted X dilution / volume plated in mls.

( Be sure to check that the colonies are V. harveyi by taking them into the dark. They

should glow)

How many bacterial cultures did you find on each plate. (TMTC = too many to count)

Calculate the concentration of bacteria in the original culture.

Do all of your calculations for your plates agree for the original concentration? Explain why or why not?

Do you calculations agree with other lab groups? Explain why or why not?

Tri-Patental Conjugation

Overview: In nature Vibrio harveyi is luminescent. Mutant strains have been developed that are not luminescent due to defective genes. We will use the following laboratory activity to demonstrate gene transfer in bacteria.

E. coli BB1 contains a plasmid (small ring of bacterial DNA) with the gene for luminescence. This plasmid can be transferred through a sex pilus.

E. coli BB2 has a plasmid for forming cell to cell bridges or sex pili. This plasmid is not transferable.

Vibrio harveyi BB151 is not luminescent because it lacks the genes for the luminescence proteins.

• Experimental: Mix 100ul of the E. coli donor ( BB1) with 100ul of E. coli helper ( BB2) and 25ul of V. harveyi ( BB151). Puddle in the middle of an LB agar plate, swirl together and incubate UPRIGHT overnight at 30^oC.

• CONTROL: Mix 100ul of the E. coli donor (BB3 - contains plasmid pLAFR2 without

the luxCDABE genes ) with 100ul of E. coli helper (BB2) and 25ul of V. harveyi (BB151).

Swirl to mix the cultures and incubate UPRIGHT overnight at 30^oC.

• Day 2 - scrape up the mixture into an eppendorf tube with 1ml of HI broth. Resuspend by pipetting up and down with a Pasteur pipette.

• Dilute the suspensions 1:10 ² by placing 10 ul into a second tube of 1ml of HI broth.

• Dilute the suspensions 1:10⁴ by placing 10ul of the 1:10² dilution into a third tube of 1ml

HI broth.

• Spread 20ul and 100ul of the 10² and 20ul and 100ul of the 10⁴ dilutions onto two HI agar plates with 100mg/ml ampicillin and 10mg/ml tetracycline.

• You will have four control and four experimental plates.

What should grow on the control plates ?

Calculate the number of conjugated V. harveyi from the dilutions.

What was the reason for having ampicillin in the plates? tetracycline?

Do the colonies luminesce? All of them? Explain.

Cross "Feeding" of Bacterial Signalling Molecules

Overview: In nature Vibrio harveyi is luminescent. Mutant strains have been developed that are not luminescent due to defective genes. We will use the following laboratory activity to demonstrate the bacterial signalling (communication) that is involved in this system.

Vibrio harveyi BB151 - lacks the genes for luminescence enzymes and substrate.

Vibrio harveyi BB202B - lacks genes for intercellular communication in the luminescence system.

• On the bottom of an HI agar plate draw two parallel lines 1cm apart.

Use a sterile cotton swab to paint one side of the agar to the line, using an overnight culture

of V. harveyi BB151. Label this side BB151.

• Use a new sterile cotton swab to paint the other side of the agar to the second line using an

overnight culture of V. harveyi BB202B. Label this side BB202B.

• Incubate overnight at room temperature.

What do you observe the next day?

How can you explain your observations?

Design an experiment to test your hypothesis.

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