Bioinformatics |
Lab Protocol |
Malaysia University |
Malaysia Bank |
Email |
Titering Tetracycline Transducing Units
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
This protocol describes two methods for preparing E. coli for infection with bacteriophage. The bacteriophage particles in this protocol carry a gene that confers tetracycline resistance. The titer of the bacteriophage particles can be determined by infecting host cells and selecting for bacteria resistant to tetracycline. Transduction is not a highly reproducible method of quantifying phage.
Procedure
A. Preparation of Starved Cells (see Hint #2)
1. Inoculate a 20 ml culture of NZY Medium with 20 μl of an overnight culture of K91BluKan and incubate at 37°C with vigorous shaking (see Hint #3).
2. When the culture reaches an optical density at 600 nm (OD600) of approximately 0.45, reduce the speed of the shaker to ≤*100 rpm for 5 min (see Hint #4).
3. Pellet the cells by centrifugation in sterile centrifuge tubes at 2200 rpm (600 X g) for 10 min in a Sorvall™ SS-34 rotor at room temperature or 4°C (see Hint #5).
4. Aspirate the supernatant and gently resuspend the cells in 20 ml of 80 mM NaCl.
5. Transfer the cell suspension to a 125-ml culture flask and shake the culture at approximately 150 to 200 rpm at 37°C.
6. Repeat Step #A3 to pellet the cells.
7. Aspirate the supernatant and gently resuspend the cell pellet in 1 ml of ice-cold NAP Buffer. Store the cell suspension at 4°C until needed (see Hint #6).
B. Preparation of Cells Grown in Terrific Broth
1. On the night before infection, inoculate 3 ml of NZY Medium containing 100 μg/ml Kanamycin from a single colony of K91BluKan (see Hint #3).
2. On the next morning, inoculate 10 ml of Terrific Broth with 100 μl of the overnight culture and grow the 10 ml culture with vigorous shaking (300 rpm) at 37°C.
3. Visually inspect the culture a few hours after inoculation. When the culture becomes turbid, measure the OD600 of 1:10 dilutions of the culture. As soon as the diluted OD600 of the 1:10 dilution reaches from between 0.125 and 0.25, reduce the speed of the shaker to ≤*100 rpm for 5 min (see Hint #4).
4. Prepare the cells as described in Steps #A6 and A7. The concentration of viable cells will be approximately 5 X 109 cells/ml.
C. Assessment of Transduction Efficiency by Measuring Tetracycline Resistance
1. Prepare serial dilutions of bacteriophage in TBS/Gelatin. If an approximate titer is known, include a range of virion particle concentrations between 3 X 105 to 3 X 106 (see Hint #7).
2. Pipette 10 μl of each serial dilution from the preceding Step into a separate plastic culture tube. Tilt the tubes approximately 10° from vertical and deposit the 10 μl sample as a droplet on the inner wall near the opening of the tube (see Hint #8).
3. Add either 10 μl of starved cells from Step #A7 or 10 μl of cells from Step #B7 to each virion dilution sample. Deposit the cells directly into the droplet of phage to ensure thorough mixing. Next, bring the tubes to a vertical position to allow the mixtures to drop to the bottom of the tubes.
4. Allow infection to proceed by incubating the tubes for 10 min at room temperature.
5. Add 1 ml of NZY Medium plus 0.2 μg/ml Tetracycline and incubate for 20 to 40 min at 37°C (see Hint #9).
6. Spread 200 μl of each culture on plates of NZY Medium plus 40 μg/ml Tetracycline and 100 μg/ml Kanamycin, and incubate overnight at 37°C (see Hint #2). On the next day, count the number of Tetracycline-resistant colonies for each dilution and calculate Transducing Units (TU)/ml for the original phage library (see Hint #10).
Solutions
TBS/Gelatin
Store at room temperature
Autoclave 0.1 g Gelatin in 100 ml of TBS
After autoclaving, swirl to mix in the melted gelatin 
TBS
Autoclave to sterilize and store at room temperature
A 10X stock can be prepared and stored at room temperature
50 mM Tris-Cl, pH 7.5
150 mM NaCl
Tetracycline (1000X)
40 ml of 40 mg/ml Tetracycline
Mix thoroughly and store at 20°C in a tube covered with aluminum foil
Filter Sterilize
Add 40 ml of autoclaved 100%(v/v) Glycerol
0.5 M Ammonium Phosphate (NH4H2PO4)
Filter Sterilize and store at 4°C
pH to 7.0 with Ammonium Hydroxide (NH4OH) (CAUTION! See Hint #1)
Kanamycin (1000X)
100 mg/ml Kanamycin Sulfate
Adjust pH to between 6 to 8 with NaOH or HCl (CAUTION! See Hint #1)
Prepare in ddH2O
Filter sterilize and store at 4°C
NAP Buffer
Filter Sterilize and store at 4°C
pH 7.0
80 mM NaCl
50 mM Ammonium Phosphate (NH4H2PO4)
0.5 M Ammonium Phosphate (NH4H2PO4)
80 mM NaCl
0.5 M Ammonium Phosphate (NH4H2PO4)
0.5 M Ammonium Phosphate (NH4H2PO4)
Terrific Broth
Dissolve in 900 ml and autoclave to sterilize
After the solution has cooled, add 100 ml of sterile Phosphate Buffer
12 g Bacto Tryptone (Difco)
24 g Bacto Yeast Extract (Difco)
4 ml of 100% (v/v) Glycerol
NZY Medium
For solid medium, add 20 g Bacto Agar (Difco) before autoclaving and pour into plates after autoclaving
Dissolve in 1 liter water
5 g NaCl
Store at room temperature
10 g NZ Amine A (Humko Sheffield Chemical)
Autoclave to sterilize
5 g Bacto Yeast Extract (Difco)
Adjust pH to 7.5 with NaOH (CAUTION! See Hint #1)
BioReagents and Chemicals
Gelatin
NZ Amine A
Tetracycline
Glycerol
HCl
Ammonium Phosphate
Ammonium Hydroxide
NaOH
Bacto Yeast Extract
Bacto Tryptone
Sodium Chloride
Tris-Cl
Kanamycin Sulfate
Protocol Hints
1. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.
2. The bacterial strain used in this protocol, K91BluKan, is kanamycin resistant. Omit kanamycin from the media if kanamycin sensitive F+ strains (cells displaying F pili) are used.
3. The high concentration of these cells promotes adsorption of phage to the F-pilus, the rate-limiting step in infection. The author of this protocol notes that cells prepared this way can be stored at 4°C for up to 5 days, and that starved cells give somewhat more reliable titers.
4. The amount of time to reach this optical density varies with experimental conditions and must be determined empirically. The additional 5 min incubation allows sheared F pili to regenerate.
5. The author of this protocol suggests the use of sterile Oak Ridge tubes.
6. The concentration of viable cells is approximately 5 X 10 9 cells/ml; cells remain viable for infection for three to five days.
7. The ideal concentration is approximately 2 X 106 virions/ml. The physical particle concentration can be calculated by multiplying the Transducing Unit (TU) concentration by 20. To perform serial dilutions, see Protocol ID#2181.
8. For titering multiple bacteriophage samples, 24-well culture dishes can also be used.
9. The low concentration of tetracycline induces expression of the tetracycline resistance gene but does not interfere with cellular protein synthesis. Incubation with gentle shaking (150 to 200 rpm) is optional. As little as 100 μl of medium can be added instead of 1 ml if dilution with medium affects efficiency of infection. This must be determined empirically.
10. 20 μl of each sample of infected cells can also be spotted in a hexagonal array up to 19 spots per 100 cm2 plate. The plates should be dried by preincubation at 37°C at least two days before spotting.
