COWS THAT PRODUCE PHARMACEUTICALS in their milk and plants that resist pests are only two of the myriad benefits promised by genetic engineering [see "Grading the Gene Tests," by John Rennie, page 88]. Whereas endowing the genomes of cows and plants with desirable traits can be difficult, genetically manipulating bacteria is fairly straightforward. You can insert a gene for resistance to penicillin into the bacterium Escherichia coli by following the steps outlined here. Similar transformations often occur naturally, as in hospitals where antibiotic-resistant bacteria sometimes proliferate.

Because E. coli is already present in your gut, there is little to worry about. But remember to adhere to the sterile procedures described. They will ensure that you do not inadvertently ingest the stuff-and that it is E. coli, and not something else, that is blossoming on your petri dishes.

The essential trick to manipulating E. coli genetically is to get the creature's single cell to think that a foreign gene is one of its own. In this case, we will graft the penicillin-resistance gene onto a plasmid from E. coli Plastids are circular loops of DNA that exist in cells independently of chromosomes. After being altered, the plasmids are able to reenter the cell and thus help us sneak our gene into the bacterium. They then replicate within the cell and produce proteins, including the ones that will bring about our desired trait. Furthermore, plasmids are propagated through successive generations. Thus, the gene is transmitted to all offspring, conferring its resistant properties to the entire colony of E. coli grown from the altered individuals.

In this experiment, we will construct three types of recombinant plasmids, called pAMP, pKAN and pAMP/pKAN, by a process called ligation. By injecting these into E. coli, we will confer resistance for ampicillin (pAMP), for kana mycin (pKAN) or for both ampicillin and kanamycin (pAMP/pKAN) to the bacterium. To see the results of our engineering, we will then grow colonies of genetically altered E. coli on plates treated with ampicillin and kanamycin.

For the materials needed in this experiment, I suggest purchasing a DNA recombination kit. In addition, you will need an aquarium (or some container of similar volume), a Bunsen burner and a few odds and ends listed in the box in Figure 2.

First, we need to build an incubator in which to grow our E. coli colonies. A 20-gallon glass aquarium will do nicely; if you do not have one, use any container of the same volume, such as a cardboard box. Place the aquarium on its side with the open end facing you. Tape a piece of plastic to the top of the aquarium so that it drapes down, covering the open end. You will need to lift the plastic up and out of the way to work inside the aquarium.

The optimal temperature for E. coli's growth is that of the human body, 98.6 degrees Fahrenheit-not surprising, given where it lives. To warm the incubator, put in a standard incandescent lamp enclosed in a can or small pail. I needed a 75-watt bulb to heat the incubator to 92 degrees F (this is less than the optimal temperature but works fine). Start out using a low-power bulb-say, 40 watts-and measure the temperature after 12 to 24 hours; increase the wattage if necessary. If the incubator becomes hotter than 98.6 degrees F, lower the wattage-a bit cooler is better than a bit hotter. If changing the bulb does not do the job, insert a light dimmer and use it to adjust the power to the lamp and consequently the temperature.

It is a good idea to have the incubator ready when the kit arrives. Open the kit and refrigerate the culture plates upside down, along with the vials of plasmid pAMP, pKAN and calcium chloride (needed for conditioning the bacteria). Freeze the vial of ligase/ligation buffer/ ATP. The other materials can be stored at room temperature. Just before starting the experiment, wipe the incubator and the work area with a 10 percent bleach solution (made by adding one part of Clorox liquid bleach to nine parts of water) or a disinfectant, such as Lysol. Also disinfect after each session and wash your hands with an antibacterial soap both before and after. Keep your work area spotless and disinfect all the hardware, such as tubes, pipettes and transfer loops, by placing them in the bleach solution after use.

Our next step is to incubate and grow E. coli bacteria on a culture plate. The culture plates contain Luria broth, or LB, which provides nutrients on which the bacteria thrive. We will need these E coli colonies for the rest of the experiment. Light the alcohol lamp or Bunsen burner. Take one culture plate labeled "LB" and mark on its bottom "E. coli" Write the date on it, too.

Select the inoculating wire loop from the kit and sterilize it by putting it into the flame of the lamp or the burner. Allow the wire to get red-hot, then remove it from the flame and hold it for a few seconds to cool. Do not put the loop down-that will contaminate it.

Place the vial of E. coli culture in your other hand and remove its cap with the little finger of the hand holding the inoculating loop. With the cap removed, briefly pass the mouth of the vial through the flame to sterilize it.

Lift the top of the LB plate marked "E. coli" just enough to insert the inoculating loop. Push the loop into the side of the jelly on the plate to cool it. Next drag the loop a few times through the E. coli culture in the vial. Remove the loop, pass the mouth of the vial through the flame again and recap it.

Drag the loop across the jellylike surface, making a Z shape in one quadrant of the plate. Replace the lid.

Turn the culture plate 90 degrees, reheat the loop and cool it by stabbing at the gel away from the first streak. Then pass the loop once through the first streak and make another zigzag shape. Repeat the turning, reflaming and streaking another two times so that there are four zigzag shapes, one in each quadrant of the culture plate. Replace the lid on the plate.

Reflame the loop before putting it down, so as not to contaminate the work space. Make this a habit.

Place the smeared plate upside down in the incubator to prevent condensation that may collect on the lid from falling into the E. coli colonies. Incubate the plate for 12 to 24 hours-no more. Then remove it from the incubator and allow the colonies to grow for one or two days at room temperature.

The next step is to link antibiotic-resistant DNA fragments with the E. coli plasmids. The vials of pAMP and pKAN contain the indicated DNA fragments as well as the plasmids; a reagent called ligase inserts the DNA fragments and reseals the plasmid loops. (ATP in the ligation solution provides energy for the joining reaction.) The procedure actually makes many different types of hybrid molecules; however, only those that are properly formed will be maintained and expressed in the cells.

Take the three vials containing 20 microliters each of ligase/ligation buffer/ ATP from the freezer. Label one vial "+pAMP/KAN," another "+pAMP" and the last one "+pKAN." Take out the tubes labeled "pAMP" and "pKAN." Using a sterile needle-nose pipette-one for each reagent-add 10 microliters of pAMP and 10 microliters of pKAN to the +pAMP/KAN vial; 10 microliters of pAMP and 10 microliters of distilled water to the +pAMP vial; and 10 microliters of pKAN and 10 microliters of distilled water to the +pKAN vial. Close the tops of the vials and gently tap their bottoms on the table to mix the reagents. Incubate the vials at room temperature for 12 to 24 hours. Now the +pAMP/pKAN vial contains plasmids for resistance to both ampicillin and kanamycin, the +pAMP vial only those for resistance to ampicillin and the +pKAN vial only those for resistance to kanamycin.

Now we must make the E. coli cells "competent" to absorb the recombinant plasmids. This requires suspending the E. coli cells in a cold calcium chloride solution and subjecting them to a brief heat shock at 107.6 degrees F. Just how DNA is absorbed by competent E. coli cells is not known.

Prepare a water bath for heat-shocking the bacteria; you will need the bath just once for 90 seconds. You can use an aquarium heater to bring the water in a container to 108 degrees F (a couple of degrees more or less should not matter ). In a pinch, just run in tap water, adjusting the temperature.

Get four sterile 15-milliliter tubes Label them "+pAMP/KAN," "-pAMP/ KAN," "+pAMP" and "+pKAN," respectively. With a sterile transfer pipette, add 250 microliters of cold calcium chloride to each tube. Place the tubes in a beaker or bowl with crushed ice.

Pick up one or two colonies of E. coli from the LB starter plate using a sterile plastic inoculating loop. Be careful not to take any gel from the plate. Immerse the loop in the calcium chloride solution in the +pAMP/KAN tube and tap against the side of the tube to dislodge the cell mass. Suspend the cells in the solution by repeatedly pipetting in and out with a sterile transfer pipette. Return the +pAMP/KAN tube to the ice. Transfer cell colonies to the other three tubes in the ice in the same way.

For each of the following steps, use a fresh needle-nose pipette. Transfer 10 microliters of ligated plasmid +pAMP/KAN from the appropriate vial to the +pAMP/KAN culture tube. Add 10 microliters of ligated +pAMP to the +pAMP culture tube and 10 microliters of ligated +pKAN to the +pKAN culture tube. Do not transfer any material into the -pAMP/KAN culture tube; this last tube should contain only unaltered E coli. Place all the tubes back in the ice and let them sit for 15 minutes.

After the ice incubation, it is time to heat-shock the E. coli cells. Remove all the tubes from the ice and immediately immerse them in the 108-degree F water bath for 90 seconds. Then return all the tubes directly to the ice. Keep them there for three to four minutes.

Using a sterile transfer pipette, add 250 microliters of Luria broth to each tube, to give your bacteria something to eat. Gently tap the tubes with your finger to mix in the broth and incubate the tubes at 98.6 degrees F for three to six hours. With luck, the E. coli should now be sufficiently shocked to absorb the plasmids in their environment.

Finally, we can check to see if our E. coli have indeed acquired resistance to ampicillin and kanamycin. Some of the culture plates in the kit already contain the antibiotics to be pitted against our E. coli and are marked as such. The plates marked "LB" contain only Luria broth; label one such plate "+LB" and the other "-LB"-on these plates we will grow the altered and unaltered E. coli, respectively. Label one LB/AMP/KAN plate "+pAMP/KAN"-to this plate, containing both ampicillin and kanamycin, we will be adding the E. coli cells resistant (we hope) to both antibiotics. Label the other LB/AMP/KAN plate "-pAMP/ KAN"-only unaltered E. coli bacteria are to be added here. Label one LB/AMP plate "+pAMP" and the other "+pKAN"- to these (ampicillin-treated) plates we will be adding E. coli resistant to ampicillin and kanamycin, respectively. Label one LB/KAN plate "+pKAN" and the other "+pAMP."

Add 100 microliters of the cell suspension from the -pAMP/KAN culture tube to the -LB plate and also to the -LB/AMP/KAN plate, using a sterile transfer pipette. Before smearing the cells over the surface of the gel, sterilize the glass spreader. Dip the spreader in the ethanol alcohol and ignite the alcohol using the Bunsen burner or alcohol lamp. After the alcohol burns off, the spreader is ready. Use it to distribute the E. coli cells evenly over the gel.

With another sterile transfer pipette- use one for each culture-add 100 microliters of the culture from the +pAMP/ KAN tube to the +LB plate and also to the +LB/AMP/KAN plate. Spread the cell suspension as before, sterilizing the glass rod each time. From the +pAMP culture tube, add 100 microliters each of the cell suspension to the +pAMP and +pKAN plates, then spread. From the +pKAN culture tube, add 100 microliters of the cell suspension to the +pAMP and +pKAN plates, then spread.

Allow the plates to gel for 10 minutes, then stack them and tape them together. Place the plates upside down in the 98.6-degree F incubator. Incubate them for 12 to 24 hours. Note that if the colonies are overincubated, they will overgrow and become indistinguishable.

Now you are ready to see the results of your experiment. On the +LB and -LB plates, both the transformed and the natural E. coli grow well. For the LB/ AMP/KAN plates-containing both ampicillin and kanamycin-there is a growth on the one labeled "+AMP/ KAN" and none on the "-AMP/KAN," showing that only the E. coli transformed with both antibiotic-resistant genes can grow. The LB/AMP plates illustrate that the gene for pAMP, and not that for pKAN, confers resistance to ampicillin. The LB/KAN plates illustrate that the gene for pKAN, and not that for pAMP, confers resistance to kanamycin.

By measuring the growth of the colonies, you can see that the ligation of two genes, pAMP and pKAN, is more difficult than a single ligation: the +pAMP/+pKAN colonies are sparser (five to 50 colonies) than are the +pAMP or the +pKAN colonies. Also, ligation Of the pKAN gene is more difficult (50 to 500 colonies) than that of the pAMP gene (100 to 1,000 colonies).

Now that you have genetically altered bacteria, you can see that in principle they are quite simple to produce. In practice, genetic engineering can be somewhat problematic. For instance, cows and plants will need much bigger incubators.

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