Gene Transfer Technique

Introduction 

Gene transfer techniques for higher plants and animals are complex and costly. It is generally not done in laboratory scale experiment. However, the techniques of gene transfer in bacteria like Escherichia coli (E. coli) are simple and appropriate for the teaching and learning laboratory. Plasmid-based genetic transformation is the most common used technology for genetic transfer. It enables manipulation of genetic information in a laboratory setting and it makes the understanding of how DNA operates. In the following protocol, we will look at the tools to transform E. coli bacteria to express new genetic information using a plasmid system and apply mathematical routines to determine transformation efficiency.

PREPARATION

Materials and Equipment

Commercial suppliers for plasmid transformation systems may be purchased in kits. These plasmids should contain the gene for ampicillin resistance (pBLU), as experimental procedures typically use ampicillin to select transformed cells. In addition, plasmids with colored marker genes like beta-GAL and fluorescence markers like green fluorescent protein (GFP) and its cousins make it possible to measure gene expression directly, to follow cell populations as they grow or move, and to find cells that have taken up a second plasmid that we cannot see easily.

The following materials are included in a typical eight-station ampicillin-resistant plasmid system.

Materials

  1. coli (1 vial or slant)
  2. Plasmid (pBLU), hydrated (20 μg)
  3. Ampicillin, lyophilized (30 μg)
  4. Transformation solution (50mM CaCl2, pH 6), sterile (15 mL)
  5. LB nutrient agar powder, sterile (to make 500 mL) (20 g) or prepared agar
  6. LB nutrient broth, sterile (10 mL)
  7. Pipettes, sterile (50)
  8. Inoculation loops, sterile (10 μL, packs of 10 loops)
  9. Petri dishes, sterile, 60 mm (packs of 20)
  10. Multicolor 2.0 mL microcentrifuge tubes (60)
  11. Microcentrifuge tube holders
  12. Clock or watch to time 50 seconds
  13. Microwave oven/water bath
  14. Thermometer that reads 42°C
  15. 1 L flask
  16. 500 mL graduated cylinder
  17. Distilled water
  18. Crushed ice and containers
  19. 10% solution household bleach
  20. Permanent marker pens
  21. Masking tape
  22. Biohazardous waste disposal bags or plastic trash bags
  23. Micropipettes, adjustable volume, 2–20 μL (and pipette tips)
  24. Parafilm laboratory sealing film
  25. 37°C incubator oven*

PREPARATION

Advance Preparation Quick Guide for Teachers

Step       Objective                                     Time Required                          When

Step 1    Prepare agar plates.                    1 hr.                                         3–7 days prior

Step 2   Rehydrate E. coli.                         2 min                                      24–36 hours prior

Streak starter plates.                   2 min                                     24–36 hours prior

Rehydrate plasmid DNA           15 min.                                   24–36 hours prior.

Step 3   Aliquot solutions.                         10 min.                                  Immediately prior

Advance Preparation for Step 1: 3–7 Days before the Transformation

  1. Prepare nutrient agar (autoclave-free).

The agar plates should be prepared at least three days before the investigation(s) are performed. Plates should be left out at room temperature for two days and then refrigerated until use. (Two days at room temperature allows the agar to cure, or dry, sufficiently to readily take up the liquid transformation solution.) If time is short, incubate the plates at 37°C overnight. This will dry them out as well, but it shortens their shelf life.  Refrigerated plates are good for up to 30 days.

To prepare the agar, add 500 mL of distilled water to a one liter or larger Erlenmeyer flask. Add the entire content of the LB nutrient agar packet. Swirl the flask to dissolve the agar and heat to boiling in a microwave or water bath or by using a hot plate with stir bar. Heat and swirl until all the agar is dissolved.

When all the agar is dissolved, allow the LB nutrient agar to cool so that the outside of the flask is just comfortable to hold (approximately 50°C.). While the agar is cooling, you can label the plates and prepare the ampicillin as outlined below in Step 3.

Pre-prepared nutrient agar also can be purchased. However, it will have to be melted before it can be poured into plates. To do this, the plastic bottles containing solid agar can be microwaved at a low temperature (such as using the “poultry defrost” option) for several minutes. Be sure to loosen the cap slightly to expel any air. At high microwave temperatures, the agar can boil over. Another option is to  place the bottles in a hot water bath; however, this will take up to 45 minutes or so to melt the agar.

  1. Prepare ampicillin.

Ampicillin is either shipped dry in a small vial or already hydrated. If shipped dry, you need to hydrate the ampicillin. Do this by adding 3 mL of transformation solution to the vial to rehydrate the antibiotic. Use a sterile pipette. The nutrient agar solidifies at 27°C, so you must be careful to monitor the cooling of the agar and then pour the plates from start to finish without interruption. Keeping the flask with liquid agar in a water bath set to 45–50°C can help prevent the agar from cooling too quickly. Before adding ampicillin to the flask of agar, make sure you can hold the flask in your bare hand (approximately 50°C). If your hand tolerates the temperature of the flask, so will the antibiotic!

  1. Label plates.

While the agar is cooling, reduce preparation time by labeling the plates. Label with a permanent marker on the bottom of each plate close to the edge. For each class using an eight-station kit, label 16 plates LB and 16 plates LB/amp.

  1. Pour nutrient agar plates.
  2. First, pour LB nutrient agar into the 16 plates that are labeled LB. If you do not do this and add ampicillin to the flask with agar, you will not be able to make control plates containing just nutrient agar.
  3. Fill each plate to about one-third to one-half (approximately 12 mL) with agar and replace the lid. You may want to stack the plates and let them cool in the stacked configuration.
  4. Add the hydrated ampicillin to the remaining LB nutrient agar. Swirl briefly to mix. Pour into the 16 plates labeled LB/amp using the same technique. Plates should set within 30 minutes.
  5. Store the plates.

After the plates have cured for two days at room temperature, they may be either used or stored by stacking them in a plastic sleeve bag slipped back down over them. The stack is then inverted, the bag taped closed, and the plates stored upside down at 4°C until used. (The plates are inverted to prevent condensation on the lid, which may drip onto the agar.)

Advance Preparation for Step 2: 24–36 Hours before the Transformation

  1. Rehydrate bacteria.

Some E. coli cultures come prepared in a slant and will not have to be rehydrated. For bacteria that must be rehydrated, use a sterile pipette to add 250 μL of transformation solution directly to the vial. Recap the vial and allow the cell suspension to stand at room temperature for 5 minutes. Then shake the mix before streaking on the LB starter plates. Store the rehydrated bacteria in the refrigerator until used (within 24 hours for best results and no longer than three days).

  1. Streak starter plates.

Starter plates are needed to produce bacterial colonies of E. coli on agar plates. Each lab team will need its own starter plate as a source of cells for transformation. LB plates should be streaked for single colonies and incubated at 37°C for 24–26 hours before the transformation investigation begins.

Using E. coli and LB agar plates, streak one starter plate to generate single colonies from a concentrated suspension of bacteria. A small amount of the bacterial suspension goes a long way. Under favorable conditions, one cell multiples to become millions of genetically identical cells in just 24 hours. There are millions of individual bacteria in a single millimeter of a bacterial colony.

  1. Insert a sterile inoculation loop straight into the vial of rehydrated bacterial culture. Remove the loop and streak the plates. Streaking takes place sequentially in four quadrants. The first streak spreads out the cells. Go back and forth with the loop about a dozen times in each of the small areas shown. In subsequent quadrants, the cells become more and more dilute, thus increasing the likelihood of producing single colonies.
  2. For subsequent streaks, use as much of the surface area of the plate as possible. After the initial streak, rotate the plate approximately 45 degrees and start the second streak. Do not dip into the rehydrated bacteria a second time. Go into the previous streak about two times and then back and forth for a total of about 10 times.
  3. Rotate the plate again and repeat streaking.
  4. Rotate the plate for the final time and make the final streak. Repeat steps a–c with the remaining LB plates for each student workstation. Although you can use the same inoculation loop for all starter plates, it is recommended that you use a new, sterile loop for each plate if you have enough. When you are finished with each plate, cover it immediately to avoid contamination.
  5. Place the plates upside down inside the incubator overnight at 37°C or at room temperature for 2–3 days if an incubator is unavailable. Use for transformation within 24–36 hours because bacteria must be actively growing to achieve high transformation efficiency. (Remember, bacterial growth is exponential.) Do not refrigerate before use. This will slow bacterial growth.
  6. coli forms off-white colonies that are uniformly circular with smooth edges. Avoid using plates with contaminant colonies such as mold.

 

  1. Prepare plasmid.

The quantity of DNA is so small that the vial may appear empty. Tap the vial or spin it in a microcentrifuge to ensure that the DNA is not sticking to the cap. If the plasmid is not hydrated, refer to instructions that come with the sample. Store the vial of hydrated DNA in a refrigerator. Rehydrated plasmid should be used within 24 hours.

 

Advance Preparation for Step 3: Immediately Before Transformation Investigation

  1. Aliquot solutions.
  2. Each student workstation will need 1 mL of transformation solution and 1 mL of LB nutrient broth. You might have to aliquot these solutions into separate color-coded 2 mL microtubes. If the LB nutrient broth is aliquoted one day prior to the lab, it should be refrigerated. Make sure to label the tubes with permanent marker.
  3. Set up the workstations. See the list of materials required. If the plasmid goes through multiple freeze-thaw cycles in a frost-free freezer, the DNA in the plasmid can degrade. It is recommended that you check the shelf life of materials with the commercial vendor.

Procedure

The plasmid likely will contain the gene for resistance to ampicillin (pBLU) antibiotic that is lethal to many bacteria, including E. coli cells. This transformation procedure involves the following three main steps to introduce the plasmid DNA into the E. coli cells and to provide an environment for the cells to express their newly acquired genes:

  1. Adding CaCl2
  2. “Heat shocking” the cells
  3. Incubating the cells in nutrient broth for a short time before plating them on agar

Materials

  1. coli starter plate prepared.
  2. Poured agar plates prepared.
  3. 2 LB agar plates
  4. 2 LB/amp agar (LB agar containing ampicillin) plates
  5. Transformation solution (CaCl2, pH 6.1) kept ice cold
  6. LB nutrient broth
  7. Sterile inoculation loops
  8. 100–1000 μL sterile bulb pipettes
  9. 1–10 μL micropipettes with sterile tips
  10. Microcentrifuge tubes
  11. Microcentrifuge tube holder/float
  12. Container full of crushed ice
  13. Marking pen
  14. DNA plasmid (0.005 μg/μL)
  15. 42°C water bath and thermometer
  16. 37°C incubator
  17. 20 μL adjustable-volume micropipettes and tips (optional)
  18. 10% household bleach
  19. Biohazardous waste disposal bags
  20. Masking or lab tape

Step 1

Label one closed microcentrifuge tube (micro test tube) “+ plasmid” and one tube “-plasmid.” Label both tubes, and place them in the microcentrifuge tube holder/float.

Step 2

Carefully open the tubes and, using a 100–1000 μL bulb pipette with a sterile tip, transfer 250 μL of the ice cold transformation solution (CaCl2) into each tube.

Step 3

Place both tubes on (into) the ice.

Step 4

Use a sterile inoculation loop to pick up a single colony of bacteria from your starter plate. Be careful not to scrape off any agar from the plate. Pick up the “+ plasmid” tube and immerse the loop into the CaCl2 solution (transforming solution) at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the solution.

Step 5

Use a new sterile 100–1,000 μL micropipette to repeatedly pulse the cells in solution to thoroughly resuspend the cells. Place the tube back on the ice.

Step 6

Using a new sterile inoculation loop, repeat Steps 5 and 6 for the “- plasmid” tube.

Step 7

Using a 1–10 μL micropipette with a sterile tip, transfer 10 μL of the plasmid solution directly into the E. coli suspension in the “+ plasmid” tube. Tap tube with a finger to mix, but avoid making bubbles in the suspension or splashing the suspension up the sides of the tube. Do not add the plasmid solution into the “- plasmid” tube.

Step 8

Incubate both tubes (“+ plasmid” and “- plasmid”) on ice for 10 minutes. Make sure the bottom of the tubes make contact with the ice.

Step 9

While the tubes are sitting on ice, label each of your agar plates on the bottom.

Step 10

Following the 10-minute incubation at 0°C, remove the tubes from the ice and “heat shock” the cells in the tubes. It is critical that the cells receive a sharp and distinct shock.Make sure the tubes are closed tightly! Place the tubes into a test tube holder/ float, and dunk the tubes into the water bath, set at 42°C, for exactly 50 seconds. Make sure to push the tubes all the way down in the holder so that the bottom of the tubes with the suspension makes contact with the warm water.

Step 11

When the 50 seconds have passed, place both tubes back on ice. For best transformation results, the change from 0°C to 42°C and then back to 0°C must be rapid. Incubate the tubes on ice for an additional two minutes.

Step 12

Remove the holder containing the tubes from the ice and place on the lab counter. Using a 100–1,000 μL micropipette with sterile tip, transfer 250 μL of LB nutrient broth to the “+ plasmid” tube. Close the tube and gently tap with your finger to mix. Repeat with a new sterile micropipette for the “- plasmid” tube.

Step 13

Incubate each tube for 10 minutes at room temperature.

Step 14

Use a 10–1,000 μL micropipette with sterile tip to transfer 100 μL of the transformation (“+ plasmid”) and control (“- plasmid”) suspensions onto the appropriate LB and LB/Amp plates. Be sure to use a separate pipette for each of the four transfers.

Step 15

Using a new sterile inoculation loop for each plate, spread the suspensions evenly around the surface of the agar by quickly “skating” the flat surface of the sterile loop back and forth across the plate surface. Do not poke or make gashes in the agar. Allow the plates to set for 10 minutes.

Step 16

Stack your plates and tape them together. Place the stack upside down in the 37°C incubator for 24 hours

Analyzing Results

By calculating transformation efficiency, you can measure the success of your transformation quantitatively.

Calculating Transformation Efficiency

Transformation efficiency = Total number of colonies growing on the agar plate/ Amount of DNA spread on the agar plate (in μg)

This quantitative measurement of transformation is the transformation efficiency. In many applications, it is important to transform as many cells as possible. For example, in some forms of gene therapy, cells are collected from the patient, transformed in the laboratory, and then put back into the patient. The more cells that are transformed to produce the needed protein, the more likely the therapy will work.

Calculating transformation efficiency gives you an indication of how effective you were in getting plasmids carrying new information into host bacterial cells. In this example, transformation efficiency is a number that represents the total number of bacterial cells that express the gene for ampicillin resistance divided by the amount of DNA plasmid used in the experiment. The transformation efficiency is calculated using the following formula.