Bac-To-Bac Baculovirus expression systems
Methods
3.4. Transposition
Prepare Luria Agar plates containing: 250ml
-50mg/ml kanamycin 250ml from 50mg/ml
-7mg/ml gentamicin 175ml from 10mg/ml
10mg/ml tetracycline 250ml from 10mg/ml
200mg/ml Bluo-gal 2.5ml from 20mg/ml
40mg/ml IPTG 50ml from 200mg/ml
Thaw the DH10Bac competent cells on ice.
Dispense50ml of cells into 15ml polypropylene tubes.
Add app. 1ng recombinant donor plasmid (in 5ml) and gently mix the DNA into the cells by tapping the side of tube.
Incubate the mixture on ice for 30min.
Heat shock the mixture by transferring to 420C water bath for 45sec
Chill the mixture on ice for 2min.
Add 900ml S.O.C. medium to the mixture.
Place the mixture in a shaking incubator at 370C with medium agitation for 4h.
Serially dilute cells using S.O.C. medium to 10-1,10-2,10-3, 10-4 (i.e. 100ml of transposition mix: 900ml of S.O.C. medium=10-1 dilution, use this to further dilute 10-fold to give 10-2 dilution, and similarly for 10-3 and 10 -4 dilution).
Place 100ml of each dilution on the plates and spread evenly over the surface.
Incubate for at least 24h at 370C (Colonies are very small and blue colonies may not be discernible prior to 24h).
3.5 Isolation of Recombinant Bacmid DNA
Select white colonies from a plate with app. 100to 200 colonies.
Note: This number facilitates differentiation between blue and white colonies.
Pick ~10 white candidates and streak to fresh plates to verify the phenotype. Incubate overnight at 370C.
From a single colony confirmed as having white phenotype on plates containing Bluo-gal and IPTG, set up a liquid culture for isolation of recombinant bacmid DNA.
The following protocol was specifically developed for isolating large plasmids (>100kb) and was adapted for isolating bacmid DNA.
Using a sterile needle, inoculate a single, isolated bacterial colony into 2ml LB medium supplemented with 50mg/ml kanamycin, 7mg/ml gentamicin, and 10mg/ml tetracycline. A 15-ml snap-cap polypropylene tube is suitable. Grow at 370C to stationary phase (up to 24h) shaking at 250 to 300rpm.
Transfer 1.5ml of culture to a 1.5-ml micro centrifuge tube and centrifuge at 14 000xg for 1min.
Remove the supernatant by vacuum aspiration and resuspend (by gently vortexing or Pipetting up and down, if necessary) each pellet in 0.3 ml of Solution I (15mM Tris-HCl pH 8.0, 10mM EDTA, 100mg/ml RNase A). Add 0.3 ml of Solution II (0.2N NaOH, 1% SDS) and gently mix. Incubate at room temperature for 5min. Note: The appearance of the suspension should change from very turbid to almost translucent.
Slowly add 0.3 ml of 3M Potassium acetate pH 5.5, mixing gently during addition. A thick white precipitate of protein and E.coli genomic DNA will form. Place the sample on ice for 5 to 10 min.
Centrifuge for 10min at 14 000 x g. During the centrifugation, label another micro centrifuge tube and add 0.8 ml absolute isopropanol to it.
Gently transfer the supernatant to the tube containing isopropanol. Avoid any white precipitate material. Mix by gently inverting tube a few times and place on ice for 5 to 10 min. At this stage, the sample can be stored at -200C overnight.
Centrifuge the sample for 15min at 14 000 x g at room temperature.
Remove the supernatant and add 0.5ml of 70% ethanol to each tube. Invert the tube several times to wash the pellet. Centrifuge for 5min at 14 000 x g at room temperature. (Optional: repeat step 8)
Remove as much of the supernatant as possible. Note: The pellet may become dislodged from the bottom of the tube, so it is better to care fully aspirate the supernatant than to pour it.
Air-dry the pellet briefly, 5 to 10 min, at room temperature and dissolve the DNA in 40ml TE. Allow the solution to sit in the tube with occasional gentle tapping of the bottom of the tube. The DNA is generally ready for use within 10min, as long as the pellets are not over dried.
Store the NA at -200C. However, avoid repeated freeze/thaw cycles to avoid a drastic reduction in transfection efficiency.
Preparations of bacmid DNA may be analyzed by agarose gel electrophoresis to confirm the presence of high molecular weight DNA.
Transfection of Sf9 Cells with Recombinant Bacmid DNA
Seed 9x105 cells per 35-mm well (of a 6-well plate) in a 2ml of Sf-900 II SFM containing penicillin/streptomycin at 0.5X final concentration (50units/ml penicillin, 50mg/ml streptomycin). Use only from a 3- to 4- day-old suspension culture in mid-log phase with viability of>97%.
Allow cells to attach at 270C for at least 1h.
Prepare the following solutions in 12 x 75-mm sterile tubes:
Solution A: For each transfection, dilute ~5ml of mini-prep bacmid DNA into 100ml Sf-900 II SFM without antibiotics.
Solution B: For each transfection, dilute~6ml CellFectin Reagent into 100ml Sf-900 II SFM without antibiotics. Note: CellFECTIN reagent is a lipid suspension that may settle with time. Mix thoroughly by inverting the tube 5-10 times before removing a sample for transfection to ensure that a homogeneous sample is taken.
Combine the two solutions, mix gently and incubate for 15 to 45 min at room temperature.
Wash cells once with 2ml of Sf-900 II SFM without antibiotics.
For each transfection, add 0.8 ml of Sf-900 II SFM to each tube containing the lipid-DNA complexes. Mix gently. Aspirate wash media from cells and overlay the diluted lipid-DNA complexes onto cells.
Incubate cells for 5h in a 270C incubator.
Remove the transfection mixtures and add 2ml of Sf-900 II SFM containing antibiotics. Incubate cells in a 270C incubator for 72h.
Harvest virus from cell culture medium at 72h post-transfection.
Harvest/Storage of Recombinant Baculovirus
When harvesting virus from the transfection, transfer the supernatant (2ml) to a sterile, capped tube. Clarify by centrifugation for 5min at 500 x g and transfer the virus containing to a fresh tube.
From the initial transfection, viral titers of 2x107 to 4x107 pfu/ml can be expected.
Store the virus at 40C, protected from light. For long tem storage of virus, the addition of fetal bovine serum (FBS) to a final concentration of at least 2%FBS is recommended. Storage of an aliquot of the viral stock at -700C is also recommended.
For determining the viral titer, a plaque assay can be performed.
Fro amplifying viral stocks, infect a suspension or monolayer culture at a Multiplicity of Infection (MOI) of 0.01 to 0.1. Use the following formula:
Innoculum required (ml): desired MOI (pfu/ml) x (total number of cells)
Titer of viral innoculum (pfu/ml)
Harvest virus at 48h post-infection. This will result app. 100-fold amplification of the virus.
Preparation of Stock Solutions
Antibiotics can be ordered in either dry powdered from or as a stabilized, sterile, premixed solution. These solutions should be stored according to the manufacturer's recommendations. Stock solutions of antibiotics dissolved in water should be sterilized by titration trough a 0.22-micron filter. Antibiotics dissolved in ethanol need not be sterilized. Store stock solutions in light-tight containers. Magnesium ions are antagonists of tetracycline. Use media without magnesium salts for selection of bacteria resistant to tetracycline.
Antibiotic Stock solution concentration Storage
Ampicillin 50mg/ml in water -200C
Kanamycin 10mg/ml in water -200C
Tetracycline 10mg/ml in ethanol -200C
Gentamicin 7mg/ml in water -200C
Bluo-gal solutions are made by dissolving the dry powder in dimethylformamide or dimethyl sulfoxide (DMSO) to make 20 mg/ml stock solution. Care must be taken when using dimethylformamide, dispense solution in a vented chemical hood only. Use glass or polypropylene tube. The tube containing the solution should be wrapped in aluminum foil to prevent damage by light and stored at -200C. It is not necessary to sterilize the solution by filtration.
A 200mg/ml stock of IPTG is made by mixing 2g of IPTG with 8ml of water until dissolved. Adjust the volume of the solution to 10ml water and sterilize by filtration through a 0.22-micron filter. Dispense the solution to 10ml with water and sterilize by filtration trough a 0.22-micron filter. Dispense the solution into several 1-ml aliquots and store at -200C.