This protocol outlines the steps to prepare competent E. coli cells harboring the pSIM plasmid for lambda Red recombination using sacB as a counter-selectable marker. The process spans three days and includes streaking, culturing, induction of lambda Red proteins, cell preparation, transformation, and selection.
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Media & Reagents:
- LB (Luria-Bertani) medium
- Appropriate antibiotic (e.g., Chloramphenicol)
- 6% Sucrose
- Autoclaved Millipore water
- Glycerol (15%)
- Pure PCR product (100-150 ng)
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Equipment:
- Incubator set to 30°C
- Shaking water baths (30-32°C and 42°C)
- Centrifuges (large and tabletop)
- Sterile centrifuge tubes (Oakwidge or 50 ml conical)
- Cold blocks or ice/water slurry
- Sterile glass pipettes and pipet tips
- Microcentrifuge tubes (1.5 ml)
- Electroporator
The protocol is divided into three main days, with each day encompassing specific tasks essential for successful lambda Red recombination.
| Step | Action | Conditions | Notes |
|---|---|---|---|
| 1 | Streak E. coli strain containing pSIM plasmid onto LB agar plates supplemented with the appropriate antibiotic. | - | Ensure proper aseptic technique to avoid contamination. |
| 2 | Incubate plates overnight at 30°C. | 30°C, Overnight | Allows for colony formation. |
| Step | Action | Conditions | Notes |
|---|---|---|---|
| 1 | Select a single colony from Day 1 plates and inoculate into 3 ml LB supplemented with the appropriate antibiotic. | 30°C, Shaking (200-250 rpm), Overnight | Use a sterile pipette or loop to transfer the colony into liquid culture. |
| 2 | Prepare autoclaved Millipore water and chill it at 4°C for use on Day 3. | - | Ensures water is sterile and cold for cell preparation steps. |
| Step | Action | Conditions | Notes |
|---|---|---|---|
| 1 | Dilute the overnight culture 1:100 into 35 ml LB with appropriate antibiotic in a 125 ml flask. | - | Example: 1 ml overnight culture into 35 ml fresh LB. |
| 2 | Incubate at 30-32°C in a shaking water bath. Monitor growth by measuring OD600. | 30-32°C, Shaking Water Bath | Ensure temperature does not exceed 32°C to maintain plasmid stability. |
| 3 | Set up an additional shaking water bath at 42°C for later use. | 42°C | Typically, use a dedicated water bath (e.g., located near the hood). |
| Step | Action | Conditions | Notes |
|---|---|---|---|
| 1 | When the culture reaches an OD600 of ~0.4, transfer 15 ml to another 125 ml flask. | - | Use a sterile transfer pipette to maintain culture integrity. |
| 2 | Incubate the transferred culture at 42°C for exactly 15 minutes to induce lambda Red protein expression. | 42°C, 15 minutes | Precise timing is crucial for optimal induction. |
| 3 | At the end of induction, place both induced and uninduced flasks into an ice/water slurry to rapidly cool the cultures. | Ice/Water Slurry, ~5 minutes | Rapid cooling halts protein expression and stabilizes cells. |
| Step | Action | Conditions | Notes |
|---|---|---|---|
| 1 | Centrifuge both cultures at 4600 x g in a large centrifuge for ~7 minutes at 4°C (or at 10,000 x g in a microcentrifuge for ~1 minute). | 4600 x g, 7 min, 4°C or 10,000 x g, 1 min, 4°C | Use pre-chilled centrifuge tubes and equipment to maintain low temperatures. |
| 2 | Carefully pour off the supernatant. Briefly blot the pellet with a paper towel without disturbing it. | - | Avoid losing the cell pellet during removal of supernatant. |
| 3 | Resuspend the cell pellet gently in ~1 ml ice-cold sterile Millipore water while keeping the tube on ice. | Ice-Cold, Sterile Water | Gentle resuspension prevents cell lysis. |
| 4 | Add 30 ml ice-cold sterile Millipore water to the resuspended cells and mix gently by inverting the tube 2 times. | - | Dilutes the cells further and aids in washing. |
| 5 | Centrifuge the mixture again under the same conditions to pellet the cells. | Same as Step 3.3.1 or 3.3.2 | Repeat the washing step to remove residual media components. |
| 6 | Pour off the supernatant, blot the pellet, and resuspend in ~1 ml ice-cold sterile Millipore water. Transfer the suspension to a cold, sterile 1.5 ml microcentrifuge tube. | - | Ensures cells are in a small volume for efficient washing. |
| 7 | Centrifuge at 10,000 x g in a microcentrifuge for ~1 minute at 4°C. | 10,000 x g, ~1 min, 4°C | Final centrifugation to concentrate cells. |
| 8 | Remove the supernatant by pipetting carefully. Keep the tube on ice. | - | Ensure minimal loss of cell pellet. |
| 9 | Resuspend the cell pellet in 200 µl ice-cold sterile Millipore water. | Ice-Cold, Sterile Water | Final resuspension step prepares cells for electroporation. |
| Step | Action | Conditions | Notes |
|---|---|---|---|
| 1 | Proceed immediately with electroporation using the prepared competent cells. | - | Freshly prepared cells have higher transformation efficiency. |
| 2 | (Optional) To store, resuspend extra cells in 15% glycerol and freeze aliquots at -80°C. Note: Recombination efficiency may be lower. | -80°C | Recommended only if immediate use is not possible. |
Below is a table detailing the setup of different experimental tubes used during the electroporation step. This includes both experimental conditions and controls essential for validating recombination events.
| Tube | Cells | DNA | Purpose/Notes |
|---|---|---|---|
| A | Induced | 100-150 ng pure PCR product | Recombination! Purpose: To facilitate the recombination event using lambda Red proteins. |
| B | Induced | None | Negative Control for Antibiotic Function and Spontaneous Resistance Purpose: To ensure that antibiotic selection is effective and to monitor for any spontaneous resistance that may arise without the presence of exogenous DNA. |
| C | Uninduced | 100-150 ng pure PCR product | Control for Plasmid DNA Contamination Purpose: To verify that any observed antibiotic resistance is due to recombination and not from contaminating plasmid DNA causing resistance without recombination. |
| D | Uninduced | None | Negative Control for Antibiotic Function and Spontaneous Resistance Purpose: Similar to Tube B, this control ensures that antibiotic selection is working correctly and helps identify any spontaneous resistance in the absence of both induction and exogenous DNA. |
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Tube A: Induced with DNA (Recombination)
- Cells: Induced (lambda Red proteins expressed)
- DNA: 100-150 ng of pure PCR product
- Purpose: This is your primary experimental setup where recombination is expected to occur. The induction ensures that lambda Red proteins are expressed, facilitating the integration of the PCR product into the bacterial genome.
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Tube B: Induced without DNA (Negative Control)
- Cells: Induced
- DNA: None
- Purpose: Serves as a negative control to confirm that any antibiotic resistance observed in Tube A is due to the recombination event and not from the induction process itself or spontaneous mutations leading to resistance.
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Tube C: Uninduced with DNA (Control for Plasmid Contamination)
- Cells: Uninduced
- DNA: 100-150 ng of pure PCR product
- Purpose: Ensures that any antibiotic resistance observed is a result of recombination rather than contamination from plasmid DNA that might confer resistance independently of the recombination process.
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Tube D: Uninduced without DNA (Negative Control)
- Cells: Uninduced
- DNA: None
- Purpose: Acts as a baseline control to monitor the inherent antibiotic resistance of the strain and to ensure that there are no spontaneous resistance events occurring in the absence of both induction and exogenous DNA.
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Prepare Electroporation Setup:
- Thaw the prepared competent cells (from Section 3.4) on ice.
- Set up your electroporation cuvettes according to the manufacturer's instructions.
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Combine Cells and DNA:
- For each tube (A, B, C, D), mix the appropriate amount of competent cells with the specified DNA (if any). Refer to the Experimental Tubes and Controls table for guidance.
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Perform Electroporation:
- Transfer the cell-DNA mixture into the electroporation cuvette.
- Electroporate under conditions optimized for your strain (commonly around 2.5 kV, 25 µF, and 200 Ω for E. coli).
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Post-Electroporation Recovery:
- Immediately transfer the contents of the cuvette to 1 ml LB (no antibiotic) in a sterile microcentrifuge tube.
- Incubate at 30-32°C with shaking for 1 hour to allow for recovery and expression of antibiotic resistance markers.
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Selection:
- Plate the transformed cells onto LB(0) with 6% sucrose and appropriate antibiotic-containing agar plates to select for recombination events and counter-selection against sacB.
- Include appropriate controls to interpret results effectively.
Integrating the Experimental Tubes and Controls is crucial for validating the success and specificity of your recombination experiments. Below is how each tube fits into the electroporation and selection workflow:
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Tube A (Induced + DNA):
- Expectation: Growth on selective media indicates successful recombination.
- Action: Analyze colonies for correct genetic modifications via PCR and sequencing.
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Tube B (Induced + No DNA):
- Expectation: Minimal to no growth; any colonies suggest antibiotic inefficacy or spontaneous resistance.
- Action: Validate antibiotic selection efficiency.
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Tube C (Uninduced + DNA):
- Expectation: Minimal to no growth; growth may indicate plasmid contamination or non-specific integration.
- Action: Confirm that antibiotic resistance is due to recombination, not plasmid contamination.
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Tube D (Uninduced + No DNA):
- Expectation: No growth; any colonies indicate inherent resistance or contamination.
- Action: Ensure that background resistance is negligible.
By comparing the growth patterns across these tubes, you can accurately assess the efficiency and specificity of your recombination events.
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Patch Testing:
- Pick approximately 20 colonies from Tube A and perform patch tests on:
- LB/cm (chloramphenicol) plates to confirm antibiotic resistance.
- LB(0) with 6% sucrose to confirm sensitivity to sacB-mediated counter-selection.
- Desired Outcome: Colonies that grow on LB(0) 6% sucrose but are not chloramphenicol-resistant indicate successful recombination.
- Pick approximately 20 colonies from Tube A and perform patch tests on:
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PCR and Sequencing:
- Perform PCR amplification of the target region in selected colonies.
- Sequence the PCR products to verify the correct genetic modifications.
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Troubleshooting:
- No Growth in Tube A: Check competence of cells, DNA quality, and electroporation conditions.
- Unexpected Growth in Control Tubes: Re-evaluate antibiotic effectiveness and check for contamination.
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Temperature Control: Maintain precise temperatures during incubation and induction steps to ensure optimal protein expression and cell viability.
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Aseptic Techniques: Always use sterile equipment and work in a clean environment (e.g., biosafety cabinet) to prevent contamination.
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Timing: Adhere strictly to incubation times, especially during the induction phase at 42°C, to avoid over- or under-induction of lambda Red proteins.
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Centrifugation: Pre-chill all centrifuges and tubes to maintain low temperatures, which is crucial for cell stability during washing steps.
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Handling Cells: Gently resuspend and wash cells to prevent cell lysis, which can decrease transformation efficiency.
This protocol can be adapted for different strains or plasmids by modifying:
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Antibiotic Selection: Adjust the antibiotic concentration based on the resistance gene present in the plasmid.
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Induction Conditions: Optimize the temperature and duration of induction based on the specific lambda Red system used.
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Volumes: Scale the volumes up or down depending on the desired number of competent cells.
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DNA Amount: Modify the amount of DNA used for transformation based on the size and complexity of the recombination event.