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Malaysia University |
Transfection of Cells with Recombinant Phage DNA by Electroporation
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
This protocol describes the preparation of electroporation-competent bacteria and their transfection with recombinant bacteriophage DNA. Preparation of a high quality phage display library depends upon a high efficiency of gene transfer into the competent cells. This procedure is similar to other protocols in the collection (see Protocol ID#551).
Procedure
A. Preparation of Frozen Electro-Competent Cells (see Hint #2)
1. Fill two 3 liter Fernbach culture flasks with 500 ml of LB per flask, cap the flasks loosely and autoclave to sterilize.
2. Inoculate a 4.5 ml NZY culture containing 100 μg/ml Streptomycin with a colony of MC1061 bacteria. Incubate the tube vertically with shaking at 300 rpm overnight at 37°C (see Hint #3).
3. Chill all solutions, tubes and equipment overnight at 4°C. Also chill a box that will hold the tubes containing electrocompetent bacteria at -80°C.
4. The next day, inoculate each flask from Step #1 with 2 ml of the overnight culture from Step #2. Grow the large cultures with shaking (300 rpm) at 37°C until they reach an optical density from between 0.4 and 0.5 at a wavelength of 600 nm (OD600).
5. While the cultures are incubating, prepare a container of crushed, dry ice powder (see Hint #4).
6. Chill the flasks for 15 min in a large pan of ice water with frequent swirling. Meanwhile, place the pre-chilled centrifuge bottles, tubes, and 0.5 ml microcentrifuge tubes on ice (see Hint #5).
7. Pour each culture evenly into three pre-chilled 250 ml centrifuge bottles and centrifuge the bottles in a Sorvall™ GSA rotor at 2000 rpm (650 X g) for 15 min at 4°C.
8. Carefully decant the supernatant without losing cells.
9. Gently resuspend the cell pellets from each bottle in 150 ml of ice-cold 1 mM HEPES, pH 7.0.
10. Repeat Steps #7 and #8.
11. Gently resuspend the cell pellets from each bottle in 60 ml of ice-cold 1 mM HEPES, pH 7.0. Combine the resuspended cells into two bottles (180 ml each) (see Hint #6).
12. Repeat Steps #7 and #8.
13. Gently resuspend the cell pellets in each bottle in 10 ml of ice-cold 10% (v/v) Glycerol. Pool the resuspended cells from both bottles into a single, pre-chilled, sterile Oak Ridge tube (see Hint #7).
14. Centrifuge the tube in a Sorvall™ SS-34 rotor at 3000 rpm (1000 X g) for 10 min at 4°C.
15. Carefully decant the supernatant.
16. Gently resuspend the cell pellet in 500 μl of ice-cold 10% (v/v) Glycerol. The total volume should be between 1.2 to 2 ml. Transfer the cell suspension to a pre-chilled 2 ml microcentrifuge tube.
17. In a refrigerated room, pipette 200 μl aliquots of the cells into pre-chilled 500 μl microcentrifuge tubes and cap the tubes securely.
18. Place each tube into dry ice such that most of the tube is immersed in the dry ice powder.
19. After 5 min, transfer the tubes to the container that has been pre-frozen in a -80°C freezer. Store the cells indefinitely at -80°C.
B. ELECTROPORATION (see Hint #8)
1. Chill 0.2 cm electroporation cuvettes and the cuvette holder of the electroporation apparatus (see Hint #9).
2. Prepare a container of crushed dry ice powder, and place the required number of tubes of frozen electro-competent cells from Step #A19 on dry ice (see Hint #10).
3. Set the electroporator controls at 2.5 kV, 25 μF and 400 Ω.
4. Assemble a rack of sterile 15 ml conical tubes (one tube per electroporation), and pipette 2 ml of SOC into each tube. Supplement the SOC with 0.2 μg/ml Tetracycline for fd-tet-derived vectors. Also gather a supply of sterile glass 5 inch Pasteur pipettes and sterile pipette tips.
5. Attach a dropper bulb on one of the Pasteur pipettes, open one of the tubes containing SOC, and place the pipette in it. Leave the cap of the open tube face-up on the bench.
6. Thaw one of the tubes of electro-competent cells by rubbing the tube back and forth between the thumb and index fingers. At the start of the thawing, open the tube of cells, add the solution of DNA, and stir gently with a pipette tip until the mixture is fully thawed. Place the tube on ice for 1 to 2 min.
7. Carefully pipette the cell/DNA mixture to the bottom of the ice-cold cuvette without introducing any air bubbles in the cuvette. Flick the cuvette to help move the suspension down to the bottom and put the cuvette back on ice for approximately 10 sec.
8. Insert the cuvette into the cuvette holder of the electroporation chamber and discharge the electrical potential of the electroporator into the cuvette (see Hint #11).
9. Upon successful completion of electrical current transfer to the cuvette, immediately add 2 ml of SOC (containing 0.2 μg/ml Tetracycline, if appropriate) to the cuvette from one of the 15 ml tubes and pipette up and down gently a few times to suspend the cells. Transfer the cell suspension back to the 15 ml tube and cap the tube loosely (see Hint #12).
10. Incubate in the tubes at 225 rpm for 1 hr at 37°C.
11. To titer transfectants and/or obtain individual transfectant clones, spread 200 μl portions of a suitable set of serial dilutions of the cultures on NZY plates containing 40 μg/ml Tetracycline (for fd-tet based vectors) and 100 μg/ml Streptomycin (for MC1061; see Hint #13).
12. For library construction, pool up to three 2 ml cultures in a single 3 liter Fernbach flask containing 1 liter of NZY Medium supplemented with 20 μg/ml Tetracycline (see Hint #14).
13. After mixing the cells with medium, remove a small sample to enable preparation of suitable serial dilutions, and spread 200 μl of these dilutions on NZY plates containing 40 μg/ml Tetracycline (for fd-tet based vectors) and 100 μg/ml Streptomycin (for MC1061). Expect a yield of 1 to 3 X 107 clones per μg DNA (see Hint #15).
Solutions
SOC
Store at room temperature
Add 1 ml of 2 M Glucose to a 100 ml bottle of SOB 
Magnesium Solution
Filter to sterilize and store at room temperature
1 M MgCl2
1 M MgSO4
0.2 M IPTG
Filter sterilize and store at 4°C
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
SOB
After cooling, add 1 ml of Magnesium Solution and Store at room temperature
0.58 g NaCl
Autoclave 100 ml portions in 125 ml bottles
Dissolve in 1 liter of ddH2O
0.19 g KCl
5 g Yeast Extract (Difco)
20 g Bacto Tryptone (Difco)
1 mM HEPES, pH 7.0 (2 liters)
9 volumes sterile ddH2O
1 volume 0.1 M HEPES, pH 7.0
Store at 4°C
2 M Glucose
Filter to sterilize
0.1 M HEPES, pH 7.0
Autoclave to sterilize and store at room temperature
Adjust pH to 7.0 with NaOH (CAUTION! See Hint #1)
100 mM HEPES
10% (v/v) Glycerol
Autoclave and store at 4°C
10% (v/v) Glycerol
Streptomycin (50 mg/ml stock)
Prepare in 10.5 ml of ddH2O
0.6 g Streptomycin Sulfate
Filter-sterilize and store at 4°C
NZY Medium
5 g NaCl
Store at room temperature
10 g NZ Amine A (Humko Sheffield Chemical)
For solid medium, add 20 g Bacto Agar (Difco) before autoclaving
Autoclave to sterilize
Dissolve in 1 liter ddH2O
5 g Bacto Yeast Extract (Difco)
Adjust pH to 7.5 with NaOH (CAUTION! See Hint #1)
LB
5 g NaCl
10 g Bacto Tryptone (Difco)
Adjust pH to 7.0 with NaOH (CAUTION! See Hint #1)
Dissolve in 1 liter ddH2O
5 g Bacto Yeast Extract (Difco)
BioReagents and Chemicals
Bacto Agar
NZ Amine A
Tetracycline
Bacto Yeast Extract
Magnesium Sulfate
Magnesium Chloride
IPTG
Sodium Chloride
Glycerol
NaOH
Streptomycin Sulfate
HEPES
KCl
Bacto Tryptone
Glucose
Protocol Hints
1. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.
2. The procedure as described will yield only half of the amount of cells required for the preparation of a large phage display library. Prepare another batch for the required amount of cells.
3. E. coli strain MC1061 is F- and has the chromosomal genotype: araD139 Δ (ara-leu)7696 Δlac174 galU galK hsr- hsm+ strAR. The strain is resistant to Streptomycin and will grow on media containing up to 100 μg/ml.
4. Liquid nitrogen can also be used in the place of dry ice; however, the contributor of this protocol did not have success using liquid nitrogen.
5. Keep the cells as cold as possible during manipulations. Keep a large bucket of ice on hand to continually chill the cell suspensions, and if possible, work in a refrigerated room. Work quickly, and treat the cells gently.
6. Gentle re-suspension of cells will increase the yield of viable competent cells. Once the cells are resuspend in the hypotonic glycerol and HEPES solutions, the cells will be vulnerable to lysis if handled roughly.
7. One can use any other tube that can be sterilized, withstand the noted g-forces during centrifugation, and are designed for use in the Sorvall™ SS-34 rotor.
8. The contributor of this protocol notes that an inexpensive alternative to the commercially available electroporation equipment can be constructed. See the following link for detailed instructions on the construction: Homemade Electroporation Apparatus.
9. These cuvettes are designed specifically for electroporation and contain metal plates for conductance of the electrical pulse. Electroporation equipment is supplied by a variety of manufacturers (e.g. the BioRad GenePulser).
10. In the preparation of large phage display libraries, the protocol contributor recommends adding up to 8 μg of DNA (in up to 15 μl of TE or ddH2O) to 200 μl of electro-competent cells. For routine small-scale electroporations, add small amounts of DNA (0.1 to 1 ng) in ≤*5 μl of buffer to 50 μl of electro-competent cells. For low concentrations of DNA, include carrier RNA at 100 μg/ml to prevent loss of DNA absorbed to vessel surfaces.
11. Please consult the manufacturer's manual for specific instructions for your equipment.
12. The low concentration of Tetracycline is not sufficient to affect sensitive cells, but is sufficient to induce expression of the Tetracycline resistance gene on fd-tet-based vectors. Thus, the successfully electroporated cells will be ready for selective growth on solid medium containing a high concentration of Tetracycline.
13. Transformation efficiencies can reach 3 X 109 clones per μg input DNA for purified RF of fd-tet-derived phage (see Protocol ID#2170).
14. If the vector is f88-4 (see Protocol ID#2176), the medium should also contain 1 mM IPTG if necessary to induce expression of the recombinant gene VIII (this is not necessary in MC1061 host cells, which do not have an intact lacI gene).
15. A 1 liter culture inoculated with three 2 ml SOC cultures (each representing 200 μl of electro-competent cells electroporated with 8 μg DNA) would yield 2.4 to 7.2 X 108 clones (2.4 to 7.2 X 105 transfectants/ml). This corresponds to 48 to 144 colonies on a plate spread with 200 μl of a 1/1000 dilution of the 1 liter culture. For a large phage display library, prepare four 1 liter cultures and titer each one independently.
Citation and/or Web Resources
1. Dower, W.J., Miller, J.F. and Ragsdale, C.W. High efficiency transformation of E. coli by high voltage electroporation. (1988) NAR 16: 6127-6145.
