A Baby Step in Creating Life

Scientists Create a Whole Genome from Scratch
February 08, 2008 Researchers have recreated a genome* from scratch. Basically they used chemistry to make 10,000 or so pieces of DNA that were about 50 nucleotides in length. Then, in a series of biochemical steps, they stuck these pieces of DNA together into around four 125,000 or so nucleotide long chunks. Then they put these chunks into yeast and let the yeast stitch together the final pieces. The end result was the reconstruction of all 582,970 nucleotides of the genome of the bacterium Mycoplasma genitalium. In the summer of last year, the same group was also able to exchange one genome for another in a bacterium. Once they do both of these in the same experiment, then they will be on the verge of creating new life. This "intelligent design" is in some ways a little scary. Scientists' track record with introducing new species has not been too successful (think rabbits in Australia). Are scientists really intelligent and wise enough to create new life? And what will the repercussions be when this technology goes mainstream? Ready or not, here new life comes. *A genome is an organism's genetic material—basically all of its DNA. For people, this would be their 23 pairs of chromosomes plus their little snippet of mitochondrial DNA.
Why this is important

Scientists can make a
genome but do not yet
fully understand how
one works.
This work showed that scientists can create pieces of DNA over half a million nucleotides long. The researchers didn't create anything new—they basically recreated a known genome. But now that they have done this, there is nothing to stop them from creating a brand new organism from scratch. Nothing but a lack of knowledge about how genomes really work that is. Scientists can make a big piece of DNA but they really don't understand how a genome works. Which makes creating totally new genomes nearly impossible. But the research done here can help them overcome this problem too. Scientists can now make new genomes with specific pieces missing and see what effect that has on the bacterium. From this scientists will gain a better understanding of how genomes work by figuring out which bits of DNA an organism needs. And which bits it doesn't. For example, scientists have been able to get rid of each of the 485 genes normally found in Mycoplasma genitalium, one at a time. Doing this they established that about 100 of them weren't essential to the bacterium's life. But what happens if they get rid of two at once? Or all 100? This kind of experiment would be very difficult to do the usual way. But if anything, it would be easier to create a smaller piece of DNA that lacks these 100 genes. Now scientists can really figure out the minimal genome necessary for life. And in the process learn how different genes work with each other to create and run an organism. Once they get a minimal genome, the next step will be to add back genes that allow the new bacterium to do what the scientists want it to do. For example, let's say they want to make a bacterium that turns plants into hydrogen to be used in fuel cells. The idea is that they would create a genome that can do this, stick it into a bacterium and ship it to the nearest plant processing center. Of course to do this, scientists need to understand what genes a bacterium needs to make hydrogen. (Scientists have yet to find or create these genes.) And once they find these genes, they need to add them in such a way that the beast can actually do what they want it to do. The end result will be a clean burning fuel made by bacteria. Why start from scratch?

There are plenty of bacteria to
choose from. Why start from
The old fashioned way of making bacteria that do new things is to mutate or add DNA back to known bacteria that already kind of do the new thing. For example, the idea would be to search for bacteria that can make hydrogen in some way. Then scientists would tweak the bacteria a bit to get them to make hydrogen from the plant source they want. This process has been pretty successful in other projects. Which begs the question of why start from scratch? Why go through all the trouble of finding and building a minimal genome? One investigator used a great analogy. Let's say you have a pile of dirt in your backyard and a hole that needs filling. And you have a car and a piece of metal. One option is to stick the metal onto the car and push the dirt to fill in the hole. The other option is to go into the garage and create a bulldozer from scratch. Why would you go into the garage and do all that work when the tools are already there? The same question can be asked with designer bacteria. Why create a genome from scratch when there are already plenty of genomes out there that, with some tweaking, can be trained to do what you want them to do? One possible advantage is versatility. Once a minimal genome is created, it should be relatively simple to add genes and test in the new bacteria. This might definitely speed up the process of mutating/creating bacteria from what is currently lying around. In our garage analogy, the idea would be that the owner sets up an automatic assembly line that can quickly create new motorized vehicles. The owner would set it up so it is easy to add pieces to create a bulldozer, tractor, or whatever else he or she needs for the yard work to get done. After the initial time lag, new machines could quickly come off the assembly line.

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How they did it

Scientists can make a
genome because A pairs
with T and G with C.
How do you make a genome from scratch? It isn't easy but it is possible because of how DNA works. As you probably know, DNA is made up of four nucleotides—A, G, C, and T. Remember too that DNA is double stranded. Basically, there is one stretch of letters opposite another one. The DNA lines up so that A is always across from T and G is always across from C. It is because of this arrangement that the investigators were able to recreate the genome. Scientists have been able to make 50 or 100 nucleotide long pieces of DNA (called oligonucleotides or oligos) for a long time. In fact, this is so routine that there are companies that will do it for you. And the investigators outsourced this part of the project to these companies. What the companies did was to create certain sized DNAs with "sticky ends." A sticky end happens when the end of a piece of DNA has only one strand of nucleotides. This single stranded DNA wants to find a partner to team up with. And the researchers provide that partner in another piece of DNA. The partner idea works because A always goes with T and G always goes with C. For example, let's say DNA 1 had a single stranded end of AATTGGCC and DNA 2 had a single stranded end of TTAACCGG. These two ends can match up to create a double stranded piece of DNA like this: ----DNA1—AATTGGCC DNA2---- ----DNA1 TTAACCGG—DNA2---- And in fact, the two ends want to match up—DNA prefers to be double stranded. Once the DNAs are partnered, the next step is to glue them together so they can't be pulled apart. The scientists did this by adding a special enzyme called ligase that basically glues together two pieces of DNA that match: ----DNA1—AATTGGCC—DNA2---- ----DNA1—TTAACCGG—DNA2---- Now you have a piece of DNA made up of DNA 1 and DNA 2. Do this a few thousand times and you end up with a whole bacterial genome. To do this and end up with a human genome, you'd need to repeat it about 120 million times. The researchers eventually made 25 pieces of DNA that were each around 24,000 nucleotides long. Then they fused three (and in one case four) of these together to make 72,000 nucleotide long pieces of DNA. Here is where they ran into a problem. In pasting together DNA, the scientists need to pass the pasted DNA through bacteria to get enough DNA for the next step in the process. In other words, once they glued DNA 1 and DNA 2 together, they needed to put it into bacteria. Then they took DNA 1,2 back out of bacteria and glued it to DNA 3,4. Then they put that into bacteria and so on. But when they tried to get the 72,000 nucleotide DNAs into bacteria, the bacteria refused. (Probably because the bacteria started to actually read all those genes and act upon them.) So the scientists switched over to a different bug to grow their DNA in—yeast. Yeast tolerated all of that DNA better than did the bacteria. So next they glued two 72,000 nucleotide DNA pieces together to make 144,000 nucleotide pieces. Then they made 290,000 nucleotide pieces and finally, they made a piece of DNA that had all 580,000 or so nucleotides. And that is how you make a small bacterium's genome from scratch. Now they have the raw material and the know how to create new genomes from scratch. Perhaps soon they'll even understand how these genomes work.