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Amplification of a Large Phage Display Library without Losing Diversity
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
This protocol describes how to amplify a phage display library without substantial loss of diversity. The library is amplified by infecting fresh cells with a portion of the library, growing the infected cells in large cultures, and isolating the phage secreted by the infected cells from medium. The key to maintaining diversity is to ensure that the number of infected cells is much larger than the number of clones in the primary library.
Procedure
1. Inoculate two 1-liter culture flasks containing 100 ml of Terrific Broth with 1 ml of an overnight culture of K91BluKan (grown in NZY containing 100 μg/ml Kanamycin). Shake the culture vigorously (300 rpm) at 37°C until the OD600 of a 1:10 dilution reaches approximately 0.2 (late log phase).
2. Slow the shaker speed (≤*100 rpm) for 5 min to allow sheared F pili to regenerate (see Hint #2).
3. Into each flask, add approximately 1012 physical particles (approximately 5 X 1010 TU) of the library to be amplified. The final concentration of physical particles (1010 virions/ml) should be roughly comparable to the concentration of viable host cells. Continue shaking the cultures slowly for 15 min.
4. Pour each culture into a separate, pre-warmed (37°C) 3-liter Fernbach flask (Bellco) containing 1 liter of NZY supplemented with 0.22 μg/ml Tetracycline (add 11 μl of 1000X Tetracycline to each liter of media). Shake the cultures vigorously (300 rpm) for 35 min at 37°C (see Hint #3).
5. To each flask, add 1 ml of Tetracycline (final concentration is 18 μg/ml).
6. Remove a 7 μl sample from each flask (see below, Step #7) and continue shaking the flasks vigorously overnight (see Hint #4).
7. Spread 200 μl of 10-4 and 10-5 serial dilutions of the two 7 μl samples from the previous step on NZY plates containing 40 μg/ml Tetracycline and 100 μg/ml Kanamycin. Count the colonies the next day. A colony count of approximately 100 on the 10-5 dilution plates indicates approximately 5 to 1010 infected cells per culture (see Hint #5).
8. Purify the virions from the cultures as described in Protocol ID#2177, omitting the detergent treatment also as described.
9. A sample of the phage library can be sequenced to confirm the degenerate region of the coding sequence.
Solutions
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 
NZY medium (1X)
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)
Phosphate Buffer
0.72 M Potassium Phosphate, Dibasic (K2HPO4)
0.17 M Potassium Phosphate, Monobasic (KH2PO4)
Autoclave to sterilize
Terrific Broth
Dissolve in 900 ml of ddH2O 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
Tetracycline (1000X)
Mix thoroughly and store at 20°C in a tube covered with aluminum foil
Filter Sterilize
40 ml of 40 mg/ml Tetracycline
Add 40 ml of autoclaved 100%(v/v) Glycerol
BioReagents and Chemicals
NZ Amine A
Potassium Phosphate, Monobasic
Tetracycline
Glycerol
Bacto Yeast Extract
Bacto Tryptone
Sodium Hydroxide
Kanamycin Sulfate
Potassium Phosphate, Dibasic
Protocol Hints
1. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.
2. Although vigorous shaking of cultures promotes rapid growth of bacteria, the delicate F pili projections are sheared. Re-growth of F pili is necessary for optimal infection with bacteriophage.
3. Fernbach flasks have three baffles at the bottom for a greater ratio of surface area to volume.
4. To create serial dilutions, the 7 μl sample is diluted in 700 μl of media. This is a 10-2 dilution of the original sample. After thorough mixing of the 10-2 dilution, dilute 7 μl from the 10-2 dilution into 700 μl of media. This is a 10-4 dilution. 70 μl of this dilution in 700 μl of media results in a 10-5 dilution.
5. For fd-tet bacteriophage, the concentration of virions per ml can be calculated by multiplying the concentration of tetracycline-resistant cells per ml by 20 (see Protocol ID#2173). Theoretically, the population of 1011 infected cells in both flasks is large enough for every clone in an initial library of one billion clones to be represented 100 times in this amplification. This is presumably sufficient over-representation to preserve essentially all the diversity of the starting library.
