Preparation and Titering of a lamda::Tn5 Bacteriophage Lysate

 

Like animals and plants, bacteria can also be infected by viruses. Such viruses are often called bacteriophage ( "bacteria eaters" ) or for short, just phage. As depicted in the diagram on the following page, the first stage of infection is adsorption. In this step a bacterial virus attaches to a specific binding site on the outer surface of the bacterial cell. By a syringe-like injection manner, the viral nucleic acid is injected into the cell, and the viral protein coat remains attached to the outside of the cell. Once the viral nucleic acid ( often DNA ) has entered the cell, replication of viral DNA and the production of viral proteins ensues. Once the component parts of the viral particle have been produced, assembly of parts occurs to produce about 100 mature bacterial viruses. Certain viral proteins then cause the break down of host cell membrane and wall, resulting in cell lysis and the liberation of the newly made virus particles. This process is called the lytic cycle and takes approximately thirty minutes. When the cell debris from this infection process is removed, what remains is called the lysate- bacteriophage in suspending fluid. The number of bacteriophage/ ml or plaque forming units/ml is called the titer.

The bacteriophage lysate that you prepare today will be for an E. coli specfic phage called lambda. It has special genetic mutations and features that we will discuss later that allow us to use this phage as an agent of mutagenesis. Of special note, this phage carries a transposon containing a kanamycin resistant gene, referred to as Tn5 within its DNA. Since the l outer surface attachment site is an E. coli protein that serves to allow the sugar maltose to pass through the outer surface membrane, host cells to be infected with l must be grown in the presence of maltose to induce the synthesis of this protein.

 

MATERIALS:

PROCEDURE:

  1. Using aseptic technique, add 0.45 ml. l dilution buffer to a sterile 13x100 sterile test tube. Label #1
  2. Using aseptic technique, add 0.9ml. l dilution buffer to six additional sterile 13x100 sterile test tubes. Label #2-7.
  3. Add .05 ml (50 ul) of the lamda::Tn5 phage stock provided to tube #1. Mix. This will result in a 1:10 or 10-1 dilution.
  4. From tube #1, remove .1 ml ( 100 ul) and add it to tube #2. Mix. This will result in a 1:100 or 10-2 dilution of the phage stock.
  5. From tube #2, remove .1 ml ( 100 ul) and add it to tube #3. Mix. This will result in a 1:1000 or 10-3 dilution of the phage stock.
  6. Continue to dilute the phage stock to 10-7.
  7. Add 0.1 ml. E. coli NK 5012 host cells to each of 5 sterile 13x100 test tubes.
  8. To each of the tubes containing host cells, add 0.1 ml. of one of the dilutions 10-3 through 10-7.
    This will yield final phage plate dilutions of 10-4, 10-5, 10-6,10-7 ,and 10-8.
  9. Incubate the host cell/ phage mixtures for 10 minutes at 37°C in a non shaking water bath.
  10. To each test tube, add 2.5 ml. of molten TB top agar. Pour each mixture atop a TB bottom agar plate. Label the bottom of each plate with the final dilution of the lysate.
  11. Add 0.1 ml. E. coli NK 5012 host cells to another sterile 13x100 test tube. Add 2.5 ml. of molten TB top agar. Pour the mixture atop a TB bottom agar plate. Label CONTROL-CELLS ONLY.
  12. Let plates solidify. Incubate at 35°C overnight.
    During this time, the lytic cycle will ensue, resulting in an exponential increase in the number of phage to ~ 109 - 1010 phage.
  13. Tomorrow in lab, you will be titering your lysate and will need a 5.0 ml. LB + 0.2% maltose + 10-3 M MgSO4 broth culture of E. coli NK5012 host cells to titer lamda::Tn 5. Aseptically, add 5.0 ml of the medium to a sterile 125 Erlenmeyer flask. Inoculate with 0.1 ml E. coli NK5012 host cells. Label and place your culture on the rotary shaker in the 35°C walk- in incubator.
  14. On the following day:
  15. To prepare the lysate, select the plate that shows nearly complete lysis. It is from this plate that the lysate will be prepared.
  16. Add 2.5 ml lamda dilution buffer to the plate surface. With a sterile glass hockey stick, "mash" the top agar layer containing the phage (lamda::Tn5) and scrape into a sterile 13x100 screw capped tube.
  17. In the hood area in Holt 307, add 4 drops of chloroform ( kept under hood in Holt 307)with a sterile pasteur pipette. Shake vigorously--simply vortexing is not sufficient. The vigorous shaking will disperse the chloroform to kill the bacterial cells and it will also help elute the phage from the top agar. Let the mixture stand at least 30 minutes at room temperature.
    NOTE: Since lamda contains only DNA and protein, it is uneffected by the choroform.
  18. Centrifuge the mixture at 5,000 rpm for 15 minutes in the table top Sorvall in Holt 303. Don't forget a balance tube!
  19. Pour off the phage containing supernatant (lysate) into another sterile 13x100 screw capped centrifuge tube. Add 1-2 drops of chloroform to the lysate. Label the tube with the phage name (host strain used), date, and your initials. Place the lysate in the refrigerator in Holt 307$deg;C until you are ready to titer it.
  20. Add several ml. water to the test tube containing the bacterial /agar debris and place in the discard basket.



Titering of lamda::Tn5 Phage Lysate by Plaque Assay

MATERIALS:

Before you begin, melt the TB top agar in the microwave according to instructor's directions.
Using a glove, place the top agar in the 45°C water bath to cool.
Retrieve your E. coli NK5012 culture from the rotary shaker in the 35°C walk-in incubator.

  1. Using aseptic technique, add 0.45 ml. lamda dilution buffer to a sterile 13x100 sterile test tube. Label #1
  2. Using aseptic technique, add 0.9ml. lamda dilution buffer to six additional sterile 13x100 sterile test tubes. Label #2-7.
  3. Add .05 ml (50 ul) of the lamda::Tn5 phage stock provided to tube #1. Mix. This will result in a 1:10 or 10-1 dilution.
  4. From tube #1, remove .1 ml ( 100 ul) and add it to tube #2. Mix. This will result in a 1:100 or 10-2 dilution of the phage stock.
  5. From tube #2, remove .1 ml ( 100 ul) and add it to tube #3. Mix. This will result in a 1:1000 or 10-3 dilution of the phage stock.
  6. Continue to dilute the phage stock to 10-7.
  7. Add 0.1 ml. E. coli NK 5012 host cells to three sterile 13x100 test tubes.
  8. To each of the tubes containing host cells, add 0.1 ml. of one of the dilutions 10-5 through 10-7.
    This will yield final phage plate dilutions of 10-6,10-7 ,and 10-8.
  9. Incubate the host cell/ phage mixtures for 10 minutes at 37°C in a non shaking water bath.
  10. To each test tube, add 2.5 ml. of molten TB top agar. Pour each mixture atop a TB bottom agar plate. Label the bottom of each plate with the final dilution of the lysate.
  11. Add 0.1 ml. E. coli NK-5012 host cells to another sterile 13x100 test tube. Add 2.5 ml. of molten TB top agar. Pour the mixture atop a TB bottom agar plate. Label CONTROL-CELLS ONLY.
  12. Let plates solidify. Incubate at 35°C overnight. Return your phage lysate to the refrigerator.
  13. On the following day:
  14. Using the colony counter, count the number of plaques on each of your plates. Record these data in tabular form and calculate the titer of the lysate (pfu [plaque forming units] /ml.). On the label of your lysate, record the titer and date. Remember to return your lysate to the refrigerator.

 


			Phage Dilution

		10-6	10-7	10-8                  

pfu





titer (pfu/ml) =

    



Transposon Mutagenesis of E. coli Cells With Suicide Vector, lamda::Tn5

Transposons are segments of double stranded DNA which are capable of moving from one genetic location to another without the aid of host homologous recombination enzymes (Rec A, Rec B and Rec C). Since they do not require sequence homology for integration into DNA, they can be used as mutagenesis tools to mutate any desired DNA sequence. (Transposons are regarded as biological mutagens in contrast to chemical mutagens like nitrosoguanidine.) If a transposon inserts within a gene, the linear continuity of that gene will be disrupted and its function will be lost.

Transposons that contain antibiotic resistance genes are very useful in genetic manipulations with bacteria. Mutants can be isolated at a high frequency even though the cells are exposed to a very low level of mutagenesis. Each mutant rarely suffers more than one mutational insertional event. Hence transposon mutagenesis avoids the problem of multiple mutations and toxicity to humans associated with chemical mutagenesis. Two of the most common transposons used in genetic manipulations are Tn5 ( encoding kanamycin resistance) and Tn10 (encoding tetracycline resistance.)

In this experiment, Tn5 will be introduced into E. coli CSH 51 by the specially constructed lamda phage, lamda::Tn5 ( lamda b221 O29 am P80am cI857(ts) rex::Tn5). This phage not only carries Tn5 but also two mutations, Oam and Pam, which prevent phage DNA synthesis in a host that does not carry an amber suppressor, and an additional mutation, b221, which blocks the ability to integrate into the E. coli chromosome. When E. coli CSH 51 cells are infected with this suicide vector, lamda::Tn5, the defective phage cannot replicate or integrate into the chromosome, but Tn5 can "hop" from the phage genome into the E. coli chromosome. This results in Kan-r cells which carry Tn5 in some random position in the chromosome. Transposition is a rare event and might occur only once per million cells infected. However, suicide vectors such as lamda::Tn5, provide powerful selection pressure for Tn5 insertions into the chromosome.

You will be selecting for E. coli CSH 51 kan-r Lac- mutants by screening cells infected with lamda::Tn5 on EMB-lactose plates containing 60 ug./ml. kanamycin. Light pink colonies will be Lac- mutants.

MATERIALS:
per pair:

for class:

PROCEDURE:

  1. Remove the two E. coli CSH 51 cultures from the 30°C shaking water bath.
  2. Add 0.2 ml of your lamda::Tn5 lysate to one culture and label "+ lamda::Tn5"
  3. Label the other culture " cells only ". This is the no phage negative control.
  4. Incubate both cultures at 30°C for 20 minutes in a non shaking water bath to allow for phage adsorption.
  5. Add 20 ml. LB broth to each culture and shake cultures at 30°C for 1 hour.
  6. Centrifuge cultures at 5,000 rpm for 10 minutes. Discard supernatant.
  7. Resuspend each cell pellet in 2.5 ml. LB broth
  8. Spread 0.l ml. per EMB- lactose + kanamycin agar plate
    • 8 plates of " cells + lamda::Tn5"
    • 1 plate of " cells only "
    • 1 plate of lamda::Tn5 lysate only (sterility control)
  9. Incubate plates at 35°C.
  10. On the following day:
    Count (or approximate ) the number of Lac+ and Lac- colonies on the EMB lactose + 60 ug/ml. kanamycin plates. (Don't forget to include those Lac- that you have picked.) Calculate the frequency of Lac- mutants.



Antibiotic Susceptibility Testing

MATERIALS:
(work in pairs)

BACKGROUND:

Antibiotics are antimicrobial agents, many of which are used to combat a bacterial infection. Different genera, and indeed even different species and strains may differ in susceptibility to various antibiotics. Therefore, before treating an infection with an antibiotic it is important to know the antibiotic susceptibility pattern (antibiogram) of the causative agent of the disease.

Several methods are used for the determination the antibiotic susceptibility. The most common method is the standardized Kirby-Bauer agar diffusion technique. This technique has the advantage of predicting the clinical effectiveness of a given antibiotic with reasonable accuracy by noting the exact diameter of the zones of bacterial growth inhibition and comparing the data with a standard table of values. The table categorizes the organism in question as susceptible, intermediate or resistant. The mere existence of a zone of inhibition does not denote that the antibiotic in question would be of value to a patient.

PROCEDURE:

  1. Each lab partner should prepare a well mixed cell suspension of his/her bacterial isolate from nature.
  2. Inoculation - For each broth culture provided or cell suspension, moisten a sterile cotton swab in the broth culture/ cell suspension and drain the excess culture fluid on the inside of the tube. Swab the entire surface of a Mueller-Hinton agar plate.
    Allow the plate to dry for 5 minutes.
  3. Placement of the disks - After the plate has dried, dispense the antibiotic disks (P, GM, CB, CF, Str, TE) from the green dispenser, onto the surface of the plate. Gently press all disks onto the agar using sterile forceps.
  4. Invert the plates and place them into the box at the front of the lab. They will be incubated at 35°C for 18 hours. At that time they will be placed into the cold room until the next laboratory period.

On the following day:

Interpretation of results:
For each organism measure the zone of inhibition in millimeters (mm) for each antibiotic and record these values on the following page. [Note: the zone of inhibition is determined by measuring the diameter of the zone of inhibition of microbial growth including the antibiotic disc.] Compare your values to the standard table and determine the antibiotic susceptibility pattern (resistant, intermediate, or susceptible) for E. coli, Staphylococcus aureus, and your bacterial soil isolate.

Organism: E. coli


	Antibiotics_Zone of inhibition (mm)		Resistant, Intermediate, or Susceptible

P 

GM 

CB 

CF 

Str

TE

Organism: Staphylococcus aureus


	Antibiotics_Zone of inhibition (mm)		Resistant, Intermediate, or Susceptible

P 

GM 

CB 

CF 

Str

TE

Organism: Your soil isolate


	Antibiotics_Zone of inhibition (mm)		Resistant, Intermediate, or Susceptible

P 

GM 

CB 

CF 

Str

TE