Regenesis (9 page)

A series of four experiments performed by Craig Venter at the J. Craig Venter Institute in Rockville, Maryland, together with a lab crew that
included the Nobel laureate Hamilton Smith, among others, illustrates the degree to which the genomes of certain organisms can be engineered, modified, whittled down, improved, and even created from scratch, chemically. In 2005 Venter and colleagues attempted to identify the essential genes of a minimal bacterium. Plenty of genes, they found, were not necessary to the organism's functioning, and some even slowed its growth.

The bacterium that Venter and his colleagues worked with was
Mycoplasma genitalium
, an organism that was already known to have the smallest genome, consisting of some 580,000 base pairs, of any known natural microbe that can be grown in pure culture. (The organism is so named because it exists as a pathogen of the human urogenital tract.)
M. genitalium
was also known to have 482 protein-coding genes. The researchers proceeded by selectively disrupting the action of various of these protein-encoding genes, one by one, and observing the effect, if any, on the bacterium. (They did not disrupt any of the forty-three RNA-encoding genes.) By this method they found that one hundred of the protein-encoding genes, or about 20 percent of the total, were nonessential, or as they called them, “dispensable.” They also found, possibly surprising to some, that disrupting some genes speeded up the organism's growth rate under certain conditions, meaning that their presence in the organism retarded its growth rate, acting as “some sort of brake on cell growth.”

The team members summed up their results: “Under our laboratory conditions, we identified 100 nonessential genes. Logically, the remaining 382
M. genitalium
protein-encoding genes, 3 phosphate transporter genes, and 43 RNA-coding genes presumably constitute the set of genes essential for growth of this minimal cell.” (Still, it is likely that loss of some of these nonessential genes could be lethal in combination. Cases abound where pairs of mutations, each of which is viable separately, lead to death of the organism if both mutations occur in the same genome.) Nonetheless, this experiment showed that the genomes of some bacteria are capable of being drastically changed without damage to the organism, and in some cases even have beneficial effects on their growth rate.

In 2007 Venter's team chemically synthesized the entire 582,970-base pair genome of
M. genitalium
. Since they worked at the J. Craig Venter
Institute, the scientists named their new synthetic genome
Mycoplasma genitalium
JCVI-1.0. The synthesis of this genome was an enormous technical achievement inasmuch as previously assembled synthetic genomes had been much smaller, the next-longest piece of synthetic DNA consisting of only 32,000 base pairs.

In their third experiment, Venter's crew changed one bacterial species into another one. They did this by taking the genome from one species and transferring it into members of the second species, which then turned themselves into members of the first. In this case the researchers used a natural (as opposed to a synthetic) genome, however, and the species in question were two different types of
Mycoplasma: M. mycoides
and
M. capricolum
. “These species are more convenient experimental organisms than
M. genitalium
because of their faster growth rate,” the researchers wrote in their report on the project, which was published in
Science
in 2009. (
M. genitalium
has an extremely slow growth rate.)

Although the procedure was technically complicated, it was simple enough conceptually, since what the researchers did was to isolate an
M. mycoides
genome and transplant it into wild-type
M. capricolum
recipient cells. For a while there were two different genomes residing in the same cell. Eventually the new DNA was recognized and taken up by the recipient cell, which thereupon transformed itself into an
M. mycoides
bacterium.

“Changing the software completely eliminated the old organism and created a new one,” Venter said of the experiment. This might at first glance sound like a magical changeover, but the invading genome was merely acting like a virus, taking over and transforming the cell into which it had been placed. Just like Venter's genome, a virus is software that completely eliminates the old organism and creates new ones.

Still, Venter's capping and culminating experiment was yet to come. This was to design, digitize, and then chemically assemble a 1.08-million base pair
M. mycoides
genome and boot it up inside a cell. They called this synthetic genome
M. mycoides
JCVI-syn1.0. Then they did with it exactly what they had done with the natural
M. mycoides
genome of the earlier experiment: transplant it into an
M. capricolum
recipient cell. The results were the same: the new (synthetic) genome took over the old
M. capricolum
cell and turned it into an
M. mycoides
cell.

As the researchers told the story in
Science
: “There was a complete replacement of the
M. capricolum
genome by our synthetic genome during the transplant process. . . . The cells with only the synthetic genome are self-replicating and capable of logarithmic growth.”

These developments created a minor sensation in the scientific world and a major sensation in the general media (“Scientists Create Artificial Life”). There were news reports saying that President Barack Obama had expressed unspecified “genuine concerns” about this work.

With one notable exception, however, Venter and his colleagues were quite restrained in their claims. In their report on the project, the researchers drew two general conclusions from what they had done. First, “the demonstration that our synthetic genome gives rise to transplants with the characteristics of
M. mycoides
cells implies that the DNA sequence upon which it is based is accurate enough to specify a living cell with the appropriate properties.” In other words, there are no mystic features, holdovers, or leftovers from vitalism pertaining to DNA molecules. Whether they were “natural” or “synthetic” genomes, they still controlled a cell.

Second, “this work provides a proof of principle for producing cells based on genome sequences designed in the computer. DNA sequencing of a cellular genome allows storage of the genetic instructions of life as a digital file.” The reduction of genetic instructions to a digital file delivered a knockout second blow to vitalism.

But then the scientists advanced a third claim: “We refer to such a cell controlled by a genome assembled from chemically synthesized pieces of DNA as a ‘synthetic cell,' even though the cytoplasm of the recipient cell is not synthetic.” They made it sound as if they had created an artificial life form even though a nonsynthetic, natural cell had actually given rise to the new organism.

The genome constitutes only about 1 percent of the dry weight of a cell, which means that only a tiny proportion of the cell is actually synthetic. The rest of the organism was as natural as any other ordinary cell. Indeed, Venter's synthetic genome depended on the rest of the recipient
cell's natural and native apparatus for its expression: it depended on the cell's molecular machinery of transcription, translation, and replication, its ribosomes, metabolic pathways, its energy supplies, and so on. (Although Venter was fond of saying that “the DNA software builds its own hardware,” it would be more accurate to say that the recipient cell builds whatever hardware the DNA software codes for—and only if the existing hardware is pretty close to the target hardware already.)

Building a living cell that is genuinely synthetic is one of the goals of synthetic biology. By separating what's essential to living systems from what's not, such a cell would advance our understanding of what constitutes the necessary and sufficient conditions for being alive. In addition, a synthetic cell, provided that it is also a
minimal
cell, is considered by some to be a beguiling platform for genomic engineering since its lack of extraneous or inhibitory components might improve its efficiency at turning out desired end products such as biofuels, medicines, vaccines, or green chemicals, although others say that a larger genome is better.

Further, discovering or creating a minimal organism would establish the limits of what's possible in the miniaturization of living systems. Biological minimalism can exist on two different levels. First, there is the minimal
genome
: the smallest genome that is sufficient to create, maintain, and replicate itself. Possibly such a genome could be as small as two 3-mers (three-part molecules) that come together to form a 6-mer (a 6-part molecule). Increasingly interesting genomes (of 187, 2587 and 113,000 base pairs) will be introduced soon (explained below).

On the second level there is the minimal
cell
, composed of the fewest components that can jointly carry out all the normal processes of life, including metabolism, reproduction, and evolution. As the genome grows larger, it gets steadily harder to separate the full length of the two strands in order to make new copies. The solution is to separate out only a little at a time (say a dozen base pairs out of millions) and synthesize a new strand of DNA or RNA a few base pairs at a time—with a long copy emerging from the intact double helix. This argues for a separation of information storage and factory functionalities. These functions are typically encoded in RNA and in proteins that fold up into complex machines instead of
being long rods. The ability to fold gives access to vast capabilities, and in principle this could be done simply with RNA genomes and with RNA as folded machines. But RNA has only four closely related functional groups (A, C, G, U), so the coding of another class of polymers (proteins) with vast diversity (20+ amino acids) was, perhaps, inevitable.

Back to what it's like for a cell to be a cell. Animations help us visualize how polymers are made from monomers. Often these are depicted as happening in an orderly fashion, similarly to how workers on an assembly line might pass car parts down the line “just in time” for the next sequential production step. The process, however, is hardly so orderly. In reality, the four nucleotides or twenty amino acids are randomly tried out before a single correct one is accepted, and each of these with considerably more jostling and false moves than is typically shown in animations.

Seemingly, a minimal genome would automatically produce a minimal organism, but this is by no means obvious. Some protozoa, for example, have genomes that are over one hundred times larger than the human genome, which means there is a big mismatch between the size of the genome and the size of the corresponding organism. This is largely due to the fact that large stretches of the protozoa's genome may consist of noncoding regions (sometimes called junk DNA), meaning dispensable under some circumstances. A minimal genome, however, would exclude such sequences by design and intention. Still, a cell produced by such a genome might nevertheless contain extraneous, redundant, or other inessential components. Whether a minimal genome will in fact produce a minimal cell is something that can be decided only after the fact, by experiment, not in advance, by theory. (But if “theory” means, as it often does, going from vast numbers of experiments to a new best guess, then the minimal genome will likely come from theory.)

Attempts to build a synthetic cell have not been entirely successful. In 1969, for example, three biologists at the State University of New York–Buffalo, K. W. Jeon, I. J. Lorch, and J. F. Danielli, decided to create a synthetic living organism. “After participating in a symposium on the experimental synthesis of living cells,” they wrote in their report on the project in
Science
, “we decided that we had the means to carry out the reassembly of
Amoeba
proteus
from its major components: namely, nucleus, cytoplasm, and cell membrane.”

Amoeba proteus
is a comparatively large (0.4 mm) aquatic organism that is easy to work with using tools such as micropipettes and other micromanipulators. And so the experimenters took the nucleus from one amoeba, the cytoplasm from a second, and put them together inside the evacuated cell membrane of a third. Eighty percent of the time, the new composite organism lived. “The techniques of cell reassembly appear to be sufficiently adequate so that any desired combination of cytoplasm, nucleus, and membrane can be assembled into living cells,” the researchers concluded.

Unfortunately this cobbled-together organism was not really a synthetic cell, for all the parts used were natural; only their locations had changed. It was more like reshuffling the deck than providing the players with a new set of cards.