Bacterial Transformation in Relationship to Terraforming

updated 12.29.02

Brian Rudo
http://www.redcolony.com


James S. Keener
http://www.redcolony.com

For pictures of the experiment: http://www.rudo.com/files/bacteria/


Brian and Jim
Jim Keener (left) and Brian Rudo having a little too much fun.
For thousands of years, people have been fascinated by the bright red star in the sky. The early Greeks and then the Romans each assigned it godhood and built temples of worship for their Ares and Mars. When the first primitive telescopes were invented, they were pointed at Mars. Early astronomers such as Lowell carefully mapped out the canali that they saw on the surface—a word later misconstrued to mean “canal,” which sparked a wholly different fascination with the planet. Science fiction writers in the nineteenth century made their living off the mystery of the red planet. When the first spacecraft were sent out to explore the solar system, the jungles of Venus and the desperate, struggling civilization of the Martians vanished, never to return. A new, different fascination with Mars has replaced these early, misguided efforts. Many scientists now believe that it is in the best interests of the human race to colonize Mars. They cite everything from the possibility of an asteroidal threat to global warming, as well as the possibility of indigenous life on Mars to convince their audiences. But it is impractical to settle Mars as it is now. The psychological hardships to be overcome from the strict confinement of an enclosed colony, and the slow, expensive nature of building cities on Mars outweigh many of the potential benefits of Martian colonization. The only practical way for humanity to make a New World out of our planetary neighbor is to make it like Earth itself, and the easiest way to do this is through genetically engineered bacteria.

Genetically engineered bacteria would be ideal for the job. To make Mars into Earth requires a new, thicker, and more breathable atmosphere, as well as biological material for plant life to grow, and water to fill oceans that do not exist on Mars. There are industrial solutions to these problems. They would involve changing the albedo of the planet by distributing huge quantities of black dust on the lighter areas of the planet, or detonating nuclear weapons at the polar caps to melt them, or building huge nuclear reactors on the planet simply to pump out heat. There are other, even more fanciful methods that have been discussed, but they quickly degenerate into the realm of billions of dollars and huge time scales. The construction costs alone to build the number of heat-releasing reactors are incredible. Some have suggested constructing tiny nano-machines that would use Martian materials to build more of themselves, and do the work required. But not only is this beyond our current technological reach, there is no reason why we should construct these when we already have efficient, natural factories to do the work for us. Bacteria are self-replicating, and naturally do many of the processes needed on Mars already on Earth, which in fact is what maintains the breathable level of oxygen in the atmosphere of our bountiful home. The genetic engineering techniques required for the slight modification that the hardiest of our bacteria require to survive in the harsh Martian conditions already exist, and in fact are used commonly to do other, less fantastic tasks. Indeed, they are so well known that they are done frequently in university research, as well as to mass-produce vital insulin for diabetics. The most common process is known as “transformation,” or the genetic engineering of a bacterium through the transfer of one organism’s genetic material to another. These facts, along with first-hand knowledge of the process, led me to believe that, using laboratory facilities in our high school, I would be able to use transformation to create an organism that would be able to survive on Mars and alter the environment to better support human life. It became clear to me through my work at Red Colony.com that no one had ever set out to create such an organism. Although an excellent theory, it was just that until someone had the bacteria to prove that it would work. In the late months of 2001, I proposed a project to create such an organism with high school facilities to Jim Keener. He was as enthusiastic as I was, and we began research about the process and obtained an idea of what materials and equipment we would need. We were lucky to obtain live cultures of halophilic (salt-loving) bacteria as well as some Escherichia coli, more commonly known as E. coli, from a professor at Carnegie Mellon University. E. coli is the disease-causing organism commonly found in undercooked meat, but which is critical to have in our intestinal systems to help digest food. The only remaining hurdles were to obtain the necessary equipment and to find the time and lab space to conduct our experiment.

Laboratory facilities were borrowed from our high school chemistry lab and some other vital pieces of equipment were loaned from various teachers around the school. The truly limiting factor was a lack of time to conduct our experiment, as supervision was required at all times in the lab.

Since this experiment had never been done before, we had to improvise the procedure that we were to use. It also immediately became clear that we would have to restrict our genetic engineering to a single attribute due to time constraints. This did not compromise our experiment, however, because once one modification was in place it would be a fairly simple matter of repeating the procedure with a different attribute. We chose to select for salt tolerance due to the simplicity of checking if the procedure had worked: we just had to grow the new bacteria in a salt environment. We finally decided on the proper procedure to conduct our transformation from analyzing several different laboratory procedures from universities across the United States, and chose which steps would be necessary for us to follow. One of the most important and universally found procedures was the technique for ensuring that no contamination occurred, along with ensuring the safety of the lab group and making sure nothing was lost.

To this end, we performed all our work under a Bunsen burner, which created a blanket of hot air that would stop most of the bacteria and particles floating in the air from landing on our exposed bacterial cultures. Before use, we always sterilized all of our metal equipment and even some of the other equipment used in the open flame until they glowed red-hot, to ensure that the instruments were not contaminants themselves. We also made sure to wear safety goggles, a lab apron, and sterilized rubber gloves at all times. Finally, we labeled our cultures with the contents so that there could be no confusion. The final procedure to do the transformation that we adopted was to sub-culture both types of bacteria, E. coli and the halophilic bacteria; test to ensure that the E. coli could not live on a halophile-specific plate; extract deoxyribonucleic acid (more commonly known as DNA, or the genetic material that controls all living cells) from the halophiles; and to insert the DNA into the E. coli through the use of the heat shock method.

The first step, sub-culturing, was simply to create more bacterial plates to work with, to use in transformation or to serve as a backup in case of failure. To do this, we simply used an inoculating loop to collect a bacterial cluster from our original petri dish, and spread the bacteria evenly across the surface of the new plate. The new plate was then incubated at 38 degrees Celsius overnight to ensure a viable colony.

After we had obtained extra cultures of the bacteria, we were able to test the validity of the experiment by attempting to grow E. coli on one of the halophile-specific plates, which contained very high salt concentrations. When the bacteria failed to grow, as we predicted, we moved on to the extraction of the DNA from the halophilic bacteria, which would be used later to augment the E. coli’s salt tolerance. To do this, we filled a 1.5 mL microcentrifuge tube with liquid halophiles. After carefully weighing the filled tube to the nearest ten-hundredth of a gram, we filled another tube with water with very close to the same weight. This was necessary to counterbalance the centrifuge, as without the counterbalance, the centrifuge would wobble and be damaged. After preparing both tubes, we placed them in the centrifuge for five minutes, after which we removed the liquid now spread out on the top, which was the now-useless halophile medium (called supernatant). In the bottom of the halophile tube was a pellet of halophilic bacteria. Over this we placed a small amount of a detergent which contained denaturing enzymes that would break apart the plasma membrane, or the outer wall of the cell. We then repeated the balancing process with another microcentrifuge tube. The centrifuge was run for thirty minutes after the denaturing detergent had been applied, and the liquid resulting was collected in another microcentrifuge tube, as this contained the DNA that we needed. We repeated this process until we had enough DNA, a mostly filled tube, to continue on to the last step. The final process to transform the bacteria was to insert the DNA into the E. coli. To do this we used a standard heat-shock method. This involves subjecting the bacteria to extremes in temperature along with a solution of calcium chloride, which opens pores in the cells’ outer walls and allows our DNA to enter the cells. To accomplish this, Jim and I placed a sample of E. coli in a microcentrifuge tube for use as a sealed container, and added a 0.1 M concentration solution of calcium chloride. We then added the DNA that had been extracted from the halophiles to the tube. To evenly distribute the cells throughout the resultant mixture, we shook the tube vigorously. Ice obtained from the school cafeteria cooled the tube for twenty minutes, and then the tube was swirled for a minute and thirty seconds in 38 degrees Celsius water for the upper extreme of heat. After five more minutes on the ice, we emptied the tube onto a prepared nutrient plate designed for halophiles and made sure the liquid was evenly distributed across the surface. To help ensure the viability of the resultant colony, we incubated the dish at 38 degrees Celsius and watched for signs of growth.

Although the microscope we had available to us was unable to focus on the bacterial cultures, we do have a fairly accurate idea of what occurred. Inside most bacterial cells, DNA floats in the cytoplasm, or inner sea of the bacteria, in groups called plasmids. These plasmids are copied by RNA and used in ribosomes to make proteins, or the building blocks of the cell. When the centrifuge, with the detergent, broke the cell walls of the halophiles, these plasmids floated free and were distributed with the other cellular residue according to their mass, which was how we were able to isolate them. They then floated in the open pores of the E. coli, and were copied by the E. coli’s own RNA and made into helpful proteins which were incorporated into the E. coli’s structure.

A major issue that we had been aware of from when we had begun the experiment dealt a huge blow to our hopes of creating a new strain of E. coli. Although we followed the correct procedures, the centrifuge that we had available to us could only spin at 1,000 revolutions per minute (rpms). The required spin rate to lyse, or break apart, the cell walls of the halophiles was 10,000 rpms—an order of magnitude higher. We had hoped to counter this through a lengthened time of exposure to the centrifuge, but apparently it was not enough to make up for the deficit. From what we could tell at the conclusion of the experiment was that the bacterial DNA never fully separated, and thus did not go into the E. coli as was hoped.

Luckily for us, or unluckily depending on the viewpoint, the day we were to present the experiment we discovered that growth had indeed occurred. The combination of the multitude of extra DNA and the small sample of transformed bacteria combined to make the bacterial growth rate incredibly slow, so slow that it took months to show any results. After hurriedly changing our report, we were able to present a successful result.

Although this was new and exciting when we began, by the time we had finished it was no longer novel. A group of scientists in Italy had successfully conducted our experiment, with more variables adjusted by the time we finished. They were able to finish faster due to their experience with transformations as well as the proper equipment and funds. Obviously, they had no knowledge of our attempt, but we are gratified and very pleased at the validity their success implies for our own project.

The bacteria produced in Italy may one day be used to transform an entire planet to become useful to humanity. We are on the cusp of this, and it is not something that we will forget easily.