Re-creating life in the lab
by Susanne Kassube
The work of a scientist can be a bit of a random walk. We are always moving in some general direction, but we need to allow for turns of events, to be able to react when suddenly an opportunity appears,” says Carlos Bustamante, professor in molecular and cell biology, physics, and chemistry at UC Berkeley. Bustamante has experienced many of those turns in the course of his career. Born in Peru, he initially went to medical school, but then decided to pursue an academic research career. After obtaining a master’s degree in biochemistry from San Marcos University in Lima, Peru, he moved to UC Berkeley to pursue graduate studies in biophysics under Professor Ignacio Tinoco. Bustamante is best known for his work in the single-molecule field, using optical and magnetic tweezers to manipulate molecules such as DNA and proteins, one at a time, and measure the forces they generate. In the Bustamante lab, single-molecule methods have been used to follow the step-by-step movements of molecular motors as well as the folding and unfolding events of single proteins.
More recently, the Bustamante lab has entered the field of synthetic biology and is working towards creating a living organism by furnishing mitochondria with the genes that might make them independent from their host cells. Mitochondria are energy-generating organelles that are found inside the cells of all eukaryotic organisms such as plants, animals, and humans. They are thought to be descendants of a bacterium that was engulfed by another cell at some point during the evolution of life. Since then, mitochondria have lost most of their genes and cannot live independently anymore, but rely on their host cell for survival. Research in the Bustamante lab aims at reintroducing the essential genes into mitochondria that will make them independent once again, to gain a better understanding of the minimal set of genes that constitutes life.
Here, Bustamante discusses the motivation for his research, his efforts to promote cutting-edge science in South America, as well as the implications of seeking to create life in the lab.
SK: What got you interested in science?
CB: I was interested in science very early on. When I was only 11 years old, I was already playing with rockets with my friends, and I also had a chemistry set and fell in love with the microscope. When I was about 12, I happened to open one of my father’s books, an autobiography of Ramon y Cajal, the Spanish neuroscientist. I was fascinated that he had developed a staining method for neurons [the Cajal stain], and that motivated me to develop a Carlos stain for onion skin. By playing with my chemistry set, I was able to develop a stain for the nuclei of the cells. I was very proud that I had launched my career in this fashion! But, although I was interested in science early on, I knew my parents expected me to become a physician, and I wanted them to be happy.
SK: How did your parents react when you decided to do something else and not study medicine?
CB: I did go to medical school initially, but then I took biochemistry in my third year, and this really changed my life. It was exactly what I wanted to do—to understand how things work at the molecular level. Of course I realized that if I were going to be a physician, I would have to stop thinking that way and instead solve the patients’ problems. At the end of the third year, when my friends started working in hospitals, I decided to stay in a biochemistry laboratory for the summer, and that experience convinced me that what I really wanted was to do science. My parents were very understanding, and it just happened that at the moment I was quitting medical school, the university in Lima introduced a master’s degree in biochemistry. I was able to validate all my credits, take some more courses, complete a bachelor’s degree in biology, and then enter the master’s degree program in biochemistry. It was quite an unusual student career.
SK: You have been involved a lot in science outreach, particularly in Latin America. Could you tell us more about that?
CB: It’s not likely that either myself or my colleagues who left Peru will return, now that we have already established our lives and careers in the United States or Europe. But I thought that what we could do is to create laboratories similar to our own—I call them twin labs—in Peru, and maintain an “umbilical cord” to the original laboratory here in Berkeley through collaborations. Right now, I have eight students at the university in Lima. They have an atomic force microscope and an optical tweezers instrument. People sometimes wonder why single-molecule studies, which are a very advanced technology even for the first world, should all of a sudden be done in Peru. But that’s the whole point! By establishing a twin laboratory in Peru, I am creating conditions in which things that could not happen otherwise are beginning to happen. I am doing a pilot experiment to see if we can create first-rate science in South America. I am lucky in having funding from different sources, but without that, it is a lot harder. I hope that the [Peruvian] government will contact selected scientists abroad and create a program to help them establish a laboratory in Peru at a university of their choosing to train people in their area of expertise. Within five or 10 years, we might be able to change the nature of research and technology in Peru completely. The country invested a lot in my education, and I would like to pay back something to that society.
SK: You are considered a pioneer of single-molecule biophysics. How did the single-molecule field come about and what motivated you to go into that direction?
CB: In 1981, John Hearst, professor in chemistry here at Berkeley at the time, attended a Cold Spring Harbor [Laboratory] symposium where a Japanese researcher showed that it was possible to see molecules of DNA with a fluorescence microscope by staining with ethidium bromide, a dye that becomes more fluorescent when it binds to DNA. When he came back to Berkeley, he told us all about it. I was incredibly fascinated by these experiments, but by the time the conference proceedings were published in 1982, I was leaving for New Mexico to start my own lab. All I did was photocopy the article and throw it into the trunk of my car. For the next two or three years, every time I opened the trunk, the paper reminded me of what I really wanted to do. But I was busy with many other things at the time: I was trying to get tenure, building my laboratory, getting new graduate students, building the machines, and teaching for the first time as a professor. However, when I received the Searle scholarship in 1984, I was able to buy a fluorescence microscope, and since the department was putting me up for tenure early, I decided that I could now concentrate on something that would be fun to do. When we repeated the previously published experiment, we could actually see the molecules of DNA dancing around. We were filming all of this, and while I was watching the movies, it became clear to me that I wanted to study the mobility of molecules in a gel during electrophoresis, and try to understand how DNA molecules separate when they move through a gel. During these experiments, we could see that DNA molecules were enormously elastic. From here, the next idea was just a very short jump: how about grabbing one of these molecules at the end and pulling it to determine how much force we have to apply in order to stretch the molecule? In order to do this, we attached one end of the DNA molecule to the coverslip and then a bead of glass to the other end, and used its weight to test the elasticity of the DNA. This is essentially the classical spring experiment, except that in our case the spring was a molecule of DNA and the weight was a microscopic bead of glass. We obtained the first curve of the elastic behavior of DNA that showed that the molecule of DNA was a non-linear spring. It soon became clear that we could manipulate single molecules to learn about their properties.
SK: What are the advantages of single-molecule over bulk studies? What information can be obtained that is not accessible through other methods?
CB: In a way it is a little unfair to knock down bulk methods. Most of what we have learned in chemistry and biochemistry has been done in bulk, by averaging everything that is going on in a solution over the entire population of molecules. There’s nothing wrong with that approach, except that the average may not always be a well-behaved entity. There is probably no molecule in the solution that is behaving exactly according to the average: some molecules react early, others late. Therefore, what you record when you follow a chemical reaction in bulk is a deceitfully clean signal, which chemists have agreed to call kinetics. By using bulk methods, you study the movement of the mean of the population, and therefore a lot of information is lost. You can easily see that in a population of chimpanzees, on average, each member of that population has one testicle and one ovary. This is mathematically correct, but biologically, physiologically, and experimentally, empirically incorrect. In other words, an average can lead to errors, and those errors in interpretation may hide information that is crucial. It is essentially the same with molecules. A lot of insight can be gained by observing the actual trajectory of a molecule undergoing a chemical reaction: how does a single molecule go from A to B, how does it react, and how does it interact with other molecules?
SK: As the single-molecule field matures, what are the challenges and big unanswered questions?
CB: I think the challenges are that we have to learn how to study ever more complex systems. As a biophysical technique, single-molecule studies are always better done in vitro: we want to make quantitative measurements, and in order to do so we have to have control over the variables that determine the behavior of the system. The ultimate hope is that one day, we will learn to manipulate biological molecules inside the cell. That poses a lot of problems and challenges because the cell is not a very controlled environment. You have 5,000 different chemical compounds inside a cell surrounding your molecule, so part of the problem is to figure out how to make quantitative measurements when we do not have control over 4,999 variables.
SK: More recently, you established a research focus in synthetic biology. What got you initially interested in doing that?
CB: I think that bio-scientists never get away from the awe of the living phenomenon—or at least, we shouldn’t. And I think that this is probably ultimately the most central question of biology: what leads to the living state? What makes matter organize itself to the point that it can reproduce and be capable of control and growth and obtain memory? I think it would be wonderful to be able to build a cell from scratch, but this is a very long shot. Therefore, the idea I had was to start halfway, by using a mitochondrion and re-engineering it in such a way that it re-gains the functions that it presumably lost throughout evolution. We are using the mitochondrion as a chassis, and putting genes back into it. It is not easy to do that, as we have learned during the last three or four years. It is a big idea, but it is also a tough idea, like all big ideas.
SK: So you think it will be possible to “create life” in the lab in the limited sense of the expression?
CB: Yes, I think so. I don’t know in how many years, and I don’t want to make a prediction, but I think that maybe in a hundred years, two biologists will meet here in Berkeley and one will tell the other, “We’ve recently been working with a new system we just put together, and it actually has very funny properties.” And they will not be talking about what exists in nature. They will be talking about what they have made in the laboratory. And that would make biology truly synthetic.
SK: Many people are skeptical about the idea of “playing God” for either ethical or biosafety reasons. How do you address these concerns?
CB: I am perfectly in agreement with the need to be careful from the point of biosafety. We do not want to be Dr. Frankenstein. However, I don’t share those ethical concerns because I don’t think we are playing God. I think we are just playing scientists. We are just doing what a scientist is supposed to do, which is to ask questions and try to understand. There was a point in chemistry where scientists stopped studying molecules that already existed in nature, and started making molecules that never existed before. Were they playing God?
SK: Most people would probably say no.
CB: No, they were just being chemists. And the same is true with biological systems—we are only trying to understand how they work. I think the issue of God and the issue of religion should be very personal and individual, but under no circumstances is it acceptable that we say that we do not want to know. That is against human nature. As long as we take care of the safety aspects of our experiments and observations, we are always better off knowing than not knowing.