From Jones and Bartlett, a book on Stem Cells from Dr. Ann A. Kiessling and Scott C. Anderson:

Selected Articles:

July 25, 2010

The Importance of Goo

First DNA, then Cytoplasm, then Freak Out.

In May, 2010, Craig Venter and his team at JCVI proudly announced that they had created the world's first entirely synthetic genome. Along with Ham Smith, Clyde Hutchison, Dan Gibson and a couple dozen other top-notch researchers, Venter assembled a strand of DNA piece by piece, complete with vanity license plates, and then inserted it into another bacteria to give it the spark of life. This new addition to the thick schmutz of life already blanketing the planet emerged from a computerized gene machine.

Is  it time to freak out?

Well, yes and no. Yes, it shows off some serious bioengineering chops, but no, it isn't a full Frankenstein moment ... yet. There are still a few chapters left in this story, but it's definitely picking up the pace.

DNA, of course, is the linear blueprint of life, all written with a puny alphabet of just four chemical letters. In double-stranded DNA, these letters hook up to form base pairs. String together a thousand or so base pairs in just the right sequence and you get genes, which are then processed by the cellular machinery into proteins. These are the basic building blocks needed to construct all things cellular, from gossamer cell membranes to the snotty goo contained within. In fact, DNA even contains instructions for making the molecular hammers and anvils used to pound out those other proteins – including the proteins that duplicate the DNA itself. If that sounds circular, it's because you're paying attention.

Venter and crew didn't create their DNA from scratch. There isn't a chart you can look at (yet) that lists the basic set of proteins you need to convert a bag of goo into a living, replicating cell. Instead Venter started with DNA from a primitive bacteria called a mycoplasma.  Unlike most bacteria, these bugs don't have a cell wall, so millions of years ago they jettisoned the genes needed to make one, further trimming their already scanty DNA. It is pretty much the smallest thing on the planet that can self-replicate. Among the mycoplasmas is one called mycoides that Venter liked because it was fast-growing and robust. It has a single chromosome neatly arranged in a circle with about a million base pairs coding for some 485 proteins.

That is a sobering thought in and of itself: you can thrive and reproduce with less than 500 proteins. Well, at least if you're content to be a slacker bacteria, totally dependent on the kindness of strangers – in this case, the special pampering that can only be found in a cozy animal cell.

Venter's team sequenced the entire genome of the mycoplasma, and at that very moment it became mere bytes in a computer, ready to edit. From that already stingy genome, they deleted bits that looked superfluous. Then, like kids with a freshly poured sidewalk in front of them, they were compelled to write their names in this genetic database, using their own secret code. To prove they aren't complete science geeks, they added literary quotes from James Joyce and other writers, genetic watermarks that would brand subsequent generations of their amazing concoction. And, to prove that they are geeks, they also encoded an HTML page with a link back to the Venter Institute.

Once the genetic database was vetted and approved, it was off to the laboratory to produce the actual DNA. Given four vials of nucleotides corresponding to the four letters of DNA,  a gene synthesizer can assemble and extrude strings of DNA like so much molecular pasta. Unfortunately there is a limit to this process, and even their minimal genome presented a huge challenge. The best they could reliably create was about a thousand base pairs at a whack. Ultimately, they recruited yeast cells to stitch together the synthesized chunks of DNA. Yeast cells have a repair mechanism that Venter's team commandeered to stitch the chunks together, over and over, going from 1,000 to 10,000, 100,000 and finally 1,000,000 base pairs. They were then ready to put their newly manufactured DNA into a host cell.

A naked strand of DNA is just a dumb molecule. In contrast, the cytoplasm is teeming with enzymatic activity, even in the absence of its cellular architect. At this point, if you had to pick which was more alive, you would likely cast your vote for the goo, not the DNA.

So of course, this would be the time to run into a major goo-related stumbling block. The problem stems from an ancient war between bacteria and viruses. You may think of these two as equally bad actors, but they belong to antagonistic worlds; they are more likely to attack each other than you. When a virus invades a bacterium, it tricks the bacterial machinery into replicating the viral DNA.  The hapless microbe becomes a zombie virus factory, suicidally churning out viruses at high speed until the bloated cell bursts open, spreading the contagion to its unsuspecting buddies. It's bacterial Armageddon. The next time you feel like whining about a cold, just be grateful that your head doesn't explode.

After many millennia of vicious microbial battles, some long-forgotten bacterium evolved a set of molecular shears to snip the invading virus into harmless little pieces. The scissors, called restriction enzymes, are tuned to cut the viral DNA at a spot between two specific base pairs. This is a fine plan except for one thing: the same set of base pairs is also found in the bacterial genome, and like an over-eager samurai, the enzymes commit molecular hara-kiri. To avoid this fate, the bacteria balances the sword with a shield, called a methyl group, to keep its own delicate targets safe. This is obviously a kludge, but nature loves kludges.

The problem was that the genes Venter assembled in the yeast didn't have the proper methyl groups, and when that naive DNA was placed in its new bacterial home, the host goo cut it to shreds. This unfriendly situation was finally remedied by adding purified bacterial methylases to shield those vulnerable spots.

Given that DNA gives rise to every single constituent of the cytoplasmic goo, it would seem to be the über-commander of the show. Indeed, it specifies every protein the cell needs to absorb food, metabolize it and excrete the waste. It manages all aspects of cellular life, right up to reproduction. But right at that propitious moment, something unexpected happens. The DNA takes a nap.

When a cell divides, it first needs to copy its DNA, and then pull the copies apart so they each end up presiding over their own daughter cells. But while the DNA is being copied, the nuclear code becomes largely inaccessible. If there was ever a time for a cell to need a protein master, it should be during division, a mind-boggling orchestration with dozens of players. It is probably the most complex thing a cell has to do, and yet at that point the master molecule is mostly a stage prop. In cells with a nucleus, the sleep is complete. In a bacteria, with no nucleus to hide in, the DNA may still be somewhat active. Nevertheless, the amazing feat of replication is accomplished largely without the help of the master molecule itself. How is this possible?

The solution is for the cell to plan ahead. Before replication, an exquisite molecular Rube Goldberg machine is painstakingly assembled. Every protein and enzyme that will be needed must be stockpiled. Every relevant chemical pathway is prepared, waiting for a final signal to start the cascade.

This is the importance of goo. The cell at this moment is poised with so much elaborate potential that it is beyond our best instruments to measure it, let alone emulate it. If there is any hand-waving in this story, it is here. If there is magic beyond the reach of reductionism, this may be its hiding place. But understood or not, the souped-up cell is now at a delicate tipping point.

Although Venter's team had cut and pasted together a splendid genomic blueprint, building the expectant cytoplasm was beyond them. As Bonnie Bassler of Princeton said, “They had to put their genome into a live cell with all the complex goo and ingredients to make the thing go.”

Venter was now ready to insert his shiny new DNA into a host cell. And that's when Venter thought it would be a cool idea to use a different species of mycoplasma as the host. You and I might think things were complicated enough, but it seemed perfectly natural to Venter. After all, if he picked a different species, he could see if foreign goo would be able to duplicate his custom DNA. What would the daughter cells look like, the host goo or the DNA?

Of course, the smart money was on the DNA, but it would somehow have to endear itself to a foreign cell and persuade it to produce its own set of genes. To make it easy to monitor this macabre romance, Venter added two other genes to his custom DNA. One of them would color the cell a brilliant blue. And using a time-honored genetics trick, the other gene conferred resistance to tetracycline. He was ready to go.

Compared with the exacting techniques it took to create the DNA, the rest of the experiment was decidedly low-tech. The researchers added their custom mycoides DNA to a beaker containing capricolum cells, stirred it gently and let it sit for a few hours. During this time, the researchers hoped that some of the mycoplasmas would trade in their original DNA for the new and improved model. Then they dosed the beaker with tetracycline, killing everything but those cells that had successfully swapped DNA. Only about seven cells in a million made the trade, but that was enough.

None of the original DNA was found in what survived; the takeover was complete. The foreign cytoplasm had embraced the new genome and swiftly copied it. It seemed oblivious to the bait and switch. The daughter cells acted like the DNA parent; the cellular goo had been reset. They dubbed their new life-form Synthia.

Plated out on petri dishes, Synthia grew from a series of tiny dots into large blue colonies. Written small in these vibrant blobs were bits of James Joyce and other authors. In a miniature version of the Body Snatchers, the host cell had been completely hijacked. A microbial printing press, it is now devoted to churning out endless copies of a few pithy quotes. Given time, however, we can expect these molecular witticisms to go the way of any other unnecessary gene: like the superfluous genes for the unneeded cell wall, they will slowly melt away.

Synthia is just a proof of concept. From this beachhead, Venter plans to expand to other bacteria, algae and yeast. From their basic body plan, the scientists will be able to add other functional goodies. One of the proposals is to splice in some genes so that a new creature might generate fuel from garbage or sunlight.  Or, given the darkening color of the Gulf of Mexico, perhaps a bug that eats oil and poops fish food would be nice. They are also looking for a way to generate custom vaccines quickly, trimming 99% off of the current time-consuming process. The more you think about it, the crazier it gets.  The idea of dialing up your own critters is positively intoxicating.

Venter calls it a "baby step," but he is being modest. This is a huge advance in biology and a monumental step toward demystifying life. If we can really be reduced to basic chemistry, a whole new philosophical paradigm may be needed. Certainly, some fundamentalists will be upset. Let's just hope they don't reach for the torches and pitchforks.

Religion aside, how scary is this baby step? So far, the chosen bacteria is fairly benign and only affects goats. But could something more insidious be attempted? It's possible, but not likely from Venter's lab, which is subject to constant scrutiny by multiple ethics reviewers. And as long as terrorists eschew science as a western plot, we may stay safe from that crazy quarter as well.

The scariest scenario might instead come from your kids. Anyone with a few hundred bucks can now purchase something called BioBricks from various institutional sources that let them mix and match genes. You can add your own home-grown inventions to existing bacteria to create fresh life forms that might do interesting things. This amazing breakthrough has kids dropping out of school to get some real world experience with synthetic biology. That's because colleges (excepting MIT and a handful of others) are having a hard time keeping up.

The institutions selling BioBricks are keeping it safe. If anyone wants something like anthrax parts, bells go off and an investigation ensues. However, what are we to think of teenagers creating life in a test tube? These are the same kids that, as most parents can attest, can't keep their rooms clean. On the other hand, we may be in greater danger from the life growing in their gym socks than anything they create in a Petri dish.  

So what are these pubescent mad scientists concocting? Some of the projects so far include bacterial arsenic detectors and infection inhibitors. So far, so awesome.

Hopefully, this bubble of creativity won't be popped by lawyers and legislators, as happened with chemistry sets. An entire generation has been protected from any hint of chemical danger, but the price we are paying is a lingering legacy of ignorance. It is surely better to expose ourselves to small risks than to run away in dumb fright. In fact, the less we know about those risks, the more likely they are to be inflicted upon us by others. Our obsession for safety may actually increase our vulnerability.

The same people who want to shut down the Large Hadron Collider – because it might somehow create a world-eating black hole – will surely want to squelch this research as well. But the real lesson we should have learned by now is that nature has had billions of years to concoct far worse dangers, from real black holes to deadly cholera, than most anything we can whip up in a lab. In fact, the deadliest products to emerge from bio-weapon labs are typically clones of entirely natural pathogens.

Our success as a species is not due to slinking cowardice, but to the courageous exercise of curiosity. Venter's baby step may engender a dose of the willies, and we certainly shouldn't abandon all caution, but the odds are that this research will usher in dramatic breakthroughs in biology, medicine, energy and even philosophy.

The next breakthrough will come from finally getting a grip on the goo. With hundreds of proteins in dozens of configurations, it's a complicated mess. But when we get it figured out, we'll truly be like gods, able to spark life out of mud.

Then we can freak out.

For more information on Dr. Venter's work, check out these sites:

The J. Craig Venter Institute

Synthetic Genomics

Copyright © 2000-2014 by Scott Anderson
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Here are some other suggested readings in synthetic biology: