Bioinformatics |
Lab Protocol |
Malaysia University |
Malaysia Bank |
Email |
Large-Scale Propagation of Frameshift Vectors (fUSE1, fUSE3 and fUSE5)
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
When vectors with gene-III frameshifts (fUSE1, fUSE3 and fUSE5) are propagated in male (infectible) strains like K91 or K91BluKan, infective pseudorevertants are strongly selected for and frequently dominate the culture when large volumes (1 liter) are involved. The contributor of the protocol strongly recommends propagating these vectors in a female (uninfectible) strain such as K802. These vectors are supplied by Prof. George P Smith at the University of Missouri and are produced by complementing their gene-III defect with a plasmid encoding wild-type pIII. The protocol describes a procedure for the small-scale propagation of the vector, extracting ssDNA, transfecting ssDNA into the uninfectible (F-) strain K802 and propagating the K802 cells for large-scale replicative form DNA preparation (see Protocol ID#2170). The protocol also includes a method for confirming that infective pseudorevertants have not accumulated in the large-scale K802 culture.
Procedure
A. Large-Scale Propagation of fUSE1, fUSE3 and fUSE5 Vectors.
1. Prepare starved K91 cells (see Protocol ID#2173).
2. Prepare 10-3, 10-4 and 10-5 serial dilutions of the fUSE1, fUSE3 or fUSE5 stock in TBS/Gelatin Solution (see Protocol ID#2181).
3. Titer 10 μl of each serial dilution as well as a titer control blank (TBS/Gelatin Solution) on the starved K91 cells as described in Protocol ID#2173 (see Hint #1).
4. Remove infected colonies from each K19 culture dish and inoculate each in 1.7 ml of NZY Media + TET in a 125 mm culture tube (see Hint #2).
5. Pour each culture into a 1.5 ml microcentrifuge tube and microcentrifuge briefly to pellet the cells.
6. Transfer the supernatant into a fresh microcentrifuge tube.
7. Prepare 1:100 dilutions of each prepared supernatant, and the titer control, using TBS/Gelatin Solution as the diluent.
8. Titer 10 μl of the 1:100 dilutions onto K19BluKan cells (Kantamycin resistant) as in Protocol ID#2173 (see Hint #3).
Although there are a few infective particles in the culture supernatant, there should be a non-infective form of the virions called "polyphage." The following steps extract ssDNA from these non-infective polyphage.
9. Microcentrifuge the remaining portion of the culture supernatant isolated in Step #A6 and pipette 1 ml of each culture into a new microcentrifuge tube.
10. Add 150 μl of PEG/NaCl Solution, mix by inversion and incubate at 4°C overnight.
11. Microcentrifuge at maximum speed for 30 min at room temperature (or at 4°C) and discard the supernatant.
12. Microcentrifuge, again, at maximum speed for 10 min and remove the remaining supernatant with a pipette.
13. Redissolve the pellet in 500 μl of TE Buffer.
14. Add 500 μl of Neutralized Phenol, mix well, and microcentrifuge at maximum speed to separate the phases.
15. Extract the aqueous (upper) phase to a fresh microcentrifuge tube.
16. Add 500 μl of 100% Chloroform to the aqueous phase, mix well, and microcentrifuge at maximum speed to separate the phases.
17. Extract the aqueous (upper) phase to a fresh microcentrifuge tube, and add:
50 μl of TE Buffer
40 μl of Sodium Acetate Solution
1 ml of 100% (v/v) Ethanol
18. Vortex the solution and incubate overnight at 4°C.
19. Microcentrifuge at maximum speed for 30 min at 4°C and discard the supernatant.
20. Microcentrifuge again at maximum speed for 10 min at 4°C and remove the remaining supernatant with a pipette.
21. Redissolve the ssDNA in 100 μl of TE Buffer.
22. Remove 20 μl for Section B and store the remaining solution at -20°C to use for future re-propagation of the vector.
B. Re-Propagation of Vector Isolated ssDNA
1. Add 180 μl of TE Buffer to 20 μl of the ssDNA and mix well.
2. Use the ssDNA solution to transfect 200 μl of K802 cells by Calcium Chloride Transfection (as described in Protocol ID#605).
3. Propagate individual colonies in 1 liter of NZY Media + TET in 3 liter Fernbach flasks, with vigorous shaking, overnight at 37°C.
C. Checking the K802 Culture for Infective Pseudorevertanats
1. Using sterile technique, transfer 1 ml of each 1 liter K802 culture into a sterile 1.5 ml microcentrifuge tube, and microcentrifuge at maximum speed to pellet the cells.
2. Transfer the supernatant into a fresh, sterile 1.5 ml microcentrifuge tube.
3. Prepare a 1:100 dilution of the culture supernatant (previous step) using TBS/Gelatin Solution as the diluent.
4. Titer 10 μl of the 1:100 culture dilution on K91BluKan cells as described in Protocol ID#2173 (include a control as described).
5. Spread 200 μl of each infection mixture on an NZY Plate containing 20 μg/ml Tetracycline and 100 μg/ml Kanamycin (see Hint #4).
6. Incubate the plates overnight at 37°C.
7. If there are more than approximately 50 colonies on the culture supernatant plates (indicating a titer of more than approximately 2.5 X 106 TU/ml in the undiluted culture supernatant), the recommendation of the contributing author is to inoculate a new 1 liter culture and start over.
Solutions
Kanamycin Stock Solution
Adjust the pH to 6.0 to 8.08 if necessary using 5 M NaOH or 5 M HCl
Store at 4°C
Filter sterilize
Dissolve Kanamycin Sulfate to 80 mg/ml in ddH2O 
Neutralized Phenol
Allow phases to separate and remove the aqueous (upper) phase
Equilibrate with Tris once more
Use water-saturated Phenol (CAUTION! see Hint #5)
Shake or vortex vigorously to equilibrate phases
Add one-tenth volume of 1 M Tris-HCl, pH 8.0
Use the lower phase as Neutralized Phenol
Sodium Acetate Solution
3 M Sodium Acetate, pH 6.0
5 M HCl
CAUTION! see Hint #5
TE Buffer
Autoclave and store at room temperature
10 mM Tris-HCl, pH 8.0
1 mM EDTA
5 M NaOH
CAUTION! see Hint #5
Tetracycline Stock Solution
Store at -20°C (the solution will not freeze), away from light
Mix thoroughly
When cool, combine the Glycerol and Tetracycline solutions
Final Tetracycline concentration is 20 mg/ml
Filter sterilize a 40 ml solution of 40 mg/ml Tetracycline prepared in ddH2O
In a 100 ml bottle autoclave 40 ml of Glycerol
NZY Media + TET
1X NZY Media
20 μg/ml Tetracycline (use Tetracycline Stock Solution)
NZY Media (10X)
Adjust the pH to 7.5 with 5 M NaOH
Store at room temperature
50 g NaCl
Dissolve in 10 liters of ddH2O
100 g NZ Amine A (Humko Sheffield Chemical)
Autoclave
50 g Yeast Extract
TBS/Gelatin Solution
After autoclaving, swirl to mix in the melted Gelatin
Store at room temperature
0.1 g Gelatin
Autoclave
100 ml 1X TBS Solution
TBS (10X)
Store at room temperature
500 mM Tris-HCl pH 7.5
1.5 M NaCl
PEG/NaCl Solution
116.9 g NaCl
Store at 4°C
Stir until the solutes dissolve (it may be necessary to heat to 65°C briefly to dissolve the last crystals of PEG)
475 ml ddH2O
100 g PEG 8000 (Union Carbide)
BioReagents and Chemicals
Phenol
Sodium Hydroxide
Tris-HCl
Gelatin
Yeast Extract
EDTA
NZ Amine A
Ethanol
Tetracycline
PEG 8000
Kanamycin Sulfate
Glycerol
Sodium Acetate
Sodium Chloride
Hydrochloric Acid
Chloroform
Protocol Hints
1. Do not use Kanamycin in the K91 medium since K91 cells are not resistant to the antibiotic. A titering generally results in roughly 3,000, 300 and 30 (10-3, 10-4 and 10-5, respectively) colonies per K91 Petri dish. This is approximately 15% of the expected titer, although low, the efficiency is sufficient to continue the protocol.
2. The fUSE1 infection may yield both tiny and normal colonies. As far as the contributor can ascertain, the normal colonies represent clones with the correct sequence. There does not appear to be any reason to use the tiny colonies.
3. Include a blank control (10μl of TBS/Gelatin Solution) in the analysis. Be sure to include kanamycin in the plate medium to ensure that only newly infected K91BluKan cells, not residual infected K91 cells, propagate. The number of colonies per plate should be 0 or 1 (a satisfactorily low background of pseudorevertants).
4. Be sure to include kanamycin as well as tetracycline in the medium to ensure that you only count newly-infected K91BluKan cells, rather than residual, vector-harboring K802 cells.
5. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
