Hacking the Genome (Winter, 2003-2004) -------------------------------------- By Professor L The creation of genetically modified organisms (GMOs) is now within the ability of a knowledgeable and dedicated hacker. The most common genetic modification is the insertion of genes from one organism into another. The recipient is called a "transgenic organism" and this article will give you enough information so that anyone who could pass a high school biology lab can create one. The usual 2600 article starts off with a disclaimer about how the article is for informational purposes only, and should the reader do anything illegal or dangerous, that's the reader's fault. The disclaimer in this article has to be stronger. Creating transgenic organisms has the potential to do great, possibly even catastrophic harm to the entire biosphere. Although the specific manipulations I describe in this article are safe (and often done in biology teaching labs), knowledge of the methods of genetic engineering have the potential to unleash enormous forces for good or for evil. The most likely harmful consequence of hackers making a mistake with genetic engineering is for the hackers to get sick or to make the people around them sick. Maybe really, really sick. If you are going to try these techniques, learn about safe laboratory practices and follow them. The consequences of screwing up with genetic engineering are much worse than a mere jail sentence, so treat it seriously. No kidding. If these techniques are so dangerous, why on earth would I want to tell hackers how to use them? I've thought about this long and hard before writing this article, and I have three reasons for writing. First, none of the information in this article is all that hard to find these days. Good high school biology classes teach the ideas (although they often figure out how to make it seem boring), and pretty much every community college will have a molecular biology lab class that teaches all of this information and good lab technique, too. If you think this article is cool, I would strongly encourage you to take a real lab mol bio course and get at the good stuff. My second reason is that I believe in the hacker mentality. When as a teenager I got tired of stacking tandems with my 8038-based blue box, I built an Imsai 8008, one of the first computer kits. Twenty-five years later, looking at my lab and all the scientific publications and prizes I have, even the straight world would have to admit that some hackers have made positive contributions to society. The hackers in the Homebrew Computer Club in the '70s spawned much of what would become Silicon Valley. The technologies that fascinate us have the power to create a radically different world; that is, they have the potential to be used for both awesome creation and awesome destruction. Hackers, who these days I think of as kids with a thirst for knowledge and the urge to try things for themselves, can be the ones with the powerfully creative ideas about how to use new technologies. And my third reason for writing is that corporate powers are already using these technologies very broadly, and in ways that I don't feel are doing justice to their potential. With this article, I hope to inspire people to learn about what genetic engineering can do, and to come up with superior alternatives to the profit-seeking corporate approach. How do corporations use genetically modified organisms? Chances are, you are eating them! Pretty much all processed food in America contains GMOs. Monsanto's Roundup Ready crops dominate worldwide commercial agriculture, including soybeans, corn, cotton, canola oil, and sugar. The particular genetic modification in these foods makes it possible to dump the weedkiller Roundup on the crops without killing them. It s convenient for industrial farmers and it helps keep Monsanto the world's largest seller of herbicides. Surely there must be a better use for transgenic organisms than that! I hope someone reading this article will one day invent it. Now that I have convinced you to be safety conscious and to strive to use this power for good (I did convince you, didn't I?), let s get started on the methods of how new genes are inserted into organisms. First, you will need to know a little bit of terminology. The base organism that we will be adding the genes to is called the "host." The host, like just about all organisms, can be thought of as a machine for turning environmentally available material and energy (food) into copies of itself. One of the key components of any organism is its genome, that is, its complete collection of genes. The genome contains all of the instructions for making the chemicals (mostly proteins) that do the work of transforming food into offspring. We are going to add a new gene, called the "transgene," to the host. Every organism is made up of cells (adult humans have about one trillion cells; many kinds of organisms consist of only a single cell), and each cell has its own copy of the organism's genome. Both the genome and the transgene are DNA molecules. DNA is a very long polymer, which means it is a molecule made up of a string of repeating components. In the case of DNA, the components are called nucleotides, and referred to by their one-letter abbreviations, A, C, T, and G. The human genome has about three billion nucleotides. The transgene we are going to insert is only a few thousand nucleotides. However, we are not going to learn how to insert new genes into human beings. Not only is that potentially very dangerous (and highly regulated), but inserting genes into all the cells of a multicellular organism like a mammal requires better laboratory technique than a first-time genetic engineer is going to be able to achieve. In this article, I will teach you how to put the firefly genes that are responsible for the firefly's glow into Escherichia coli (E. coli for short), the bacterium that lives in your gut. You're going to make intestinal bacteria that glow in the dark. So, in this article, the host will be E. coli and the transgenes will be the gene from fireflies that make them glow. This gene is called Luciferase. (Who says scientists don't have a sense of humor?) In order to do your genetic engineering, you will first have to learn how to grow controlled populations of bacteria. Growing bacteria is a lot like keeping any other kind of pet. You need a source of them to start with, you need a home for them that keeps them safe (mostly from other creatures or contaminants), and you need to make sure they have the right kind of food, the right temperature, and so on. Because cells are too small to see, it helps to have a microscope for this work, although it s not strictly necessary. Bacteria reproduce very quickly and when enough of them grow together (called a colony), they are visible to the naked eye. In order to get started, you need to get some E. coli, some agar-coated petri dishes (their food and home), and loop (a simple thin piece of metal for transporting cells from the source to the dish). You also will need to learn a little about sterile lab procedures so that you don't contaminate your cells. In the "Sources" section at the end of this article, I recommend a kit that you can buy pretty cheaply that has all the materials you need. Eventually, you'll know enough to be able to scrounge all kinds of cool materials for genetic engineering that cost little or nothing, but I'd recommend starting with the kit. The key task is getting the transgene into the genome of the E. coli. Hosts, of course, have various methods for resisting the addition of foreign DNA. The most basic of these is the cell membrane, which acts like skin for cells. It's the job of the membrane to keep the insides in and the outsides out. However, membranes have to let in food and let out wastes, so they are permeable. In order to get the transgene inside the cell, we have to manipulate it so that it will take up the new genes. For bacteria, figuring out this problem is really the main task in creating a transgenic organism, and it's pretty easy. For higher organisms, there is more structure (the genome stays in an internal structure called the nucleus of the cell) and better defenses against foreign DNA, making the insertion of transgenes more difficult. However, inserting transgenes into higher organisms (including mammals, like mice or monkeys) is routine laboratory procedure these days. In addition to making the E. coli take in the foreign DNA, we have to make sure that the DNA is treated as if it were the organism s own. In bacteria, this is also fairly easy. Bacteria often exchange small pieces of DNA, called plasmids, with each other. These plasmids are separate from the organism's main DNA and allow bacteria to exchange beneficial genetic material with each other, even though they don't replicate sexually. (Sex is nature's best way of exchanging genetic material between organisms.) Vector is the name that biologists use for something that can introduce foreign DNA into a cell. Plasmids are good vectors for bacterial hosts. Other vectors that work better for more complex hosts include viruses that have had transgenic payloads grafted into them, or even tiny gold beads coated with DNA that can be shot into a cell with a "gene gun." The creation of plasmids (or other vectors) with transgenic payloads is made possible by the existence of DNA splicing enzymes. Simple laboratory techniques allow the extraction of naturally occurring plasmids from bacteria and splicing the DNA for the new gene into them. The hardest part is figuring out which combination of genes to insert into a host in order to get a desired effect. However, those techniques are beyond the scope of this introductory article. For our purposes, we can just buy plasmids with our desired genes from a scientific supply house. An E. coli plasmid with the Luciferase gene in it is called pUC18-luxR, and can be purchased from many places (see "Sources" section, below). Once you have successfully grown some E. coli colonies and purchased your Luciferase plasmid, the process of creating glow-in-the-dark bacteria is pig-easy. You make the bacterial membrane permeable to the plasmid by treating it with a solution of calcium chloride. At this point, the cells are said to be "competent" for transformation and the plasmids can be added. Then let the cells grow at body temperature (37C) for 12 to 24 hours. Turn out the lights and look at your petri dish; you should be able to see colonies that quite clearly glow in the dark. Congratulations! You've just created your first transgenic organism! The recommended kit has detailed instructions (called a protocol in molecular biology). The protocol can also be downloaded from the Net without buying the kit. Now if this feels too much like the script kiddy version of genetic engineering, then there are lots of other projects you might take on. You can design and construct your own plasmids, perhaps with multiple transgenes. In order to breed pure populations of transgenic bacteria, one often includes an antibiotic resistance gene in the plasmid, and then applies the antibiotic to the petri dishes. Only the bacteria that took up the plasmid will survive, and the evolutionary selective pressure will ensure that the bacteria won't lose the transgenes. In considering which genes to add, you might learn to use GenBank and LocusLink, two important Web-accessible databases of genes. Start by looking up green fluorescent protein (GFP). Or buy a GFP transgenic fish from GloFish. Hacking the genome is the future. You can be there now.... Sources A complete kit with everything you need to do this experiment is available from Modern Biology, Inc. for less than $75. It is part number IND-9 and you can order it on the Web. Visit http://www.modernbio.com/ind-9.htm to see what's in the kit and how to order it. Modern Biology has all kinds of really cool kits that don t require fancy labs or a lot of experience to use. Check out their whole catalog at http://www.modernbio.com/ TableOContents.htm. A different $80 kit allows you to extract DNA from any organism (including yourself), which, with some DNA splicing enzymes and some additional knowledge of how to recombine bits of DNA, you could then use for creating new plasmids. It's available from the Discovery Channel store. This kit includes an inexpensive centrifuge, which you are going to need if you want to continue your genetic engineering experimentation. You can get good scientific microscopes on eBay or maybe you have one in a basement somewhere. If you're going to work with GFP, you probably want a microscope for fluorescence work; it will have a filter set and high power illumination. If you would like proof that many of the foods you eat contain genetically modified organisms, you might be interested in the kits available from Investigen, which uses a similar technology for easy detection of many genetically modified organisms. See http://www.investigen.com/products.html for the details. If you want to look up interesting genes that you might want to add to your bacteria, try using GenBank or LocusLink from http://ncbi.nlm.nih.gov. Once you get good at transforming bacteria and want to start thinking about more ambitious transgenic organisms, you should take a look at the offerings from Clontech, Qiagen, and Qbiogene. Or you can just buy a GFP zebrafish from http://www.glofish.com. And before you start working on your plan for creating a Luciferase transgenic puppy by doing genetic engineering on your dog, you should probably learn real molecular biology laboratory techniques by taking a class. Who knows, maybe I'll be your teacher... Shoutouts: DMcS for taking it seriously and finding the GloFish and the Discovery kit, and to AG Monster for reminding me that although I am old now, I was a hacker once, too.