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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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July 6, 2011

A First Step Toward A New Form of Life

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Posted by Derek

There's been a real advance in the field of engineered "unnatural life", but it hasn't produced one-hundredth the headlines that the arsenic bacteria story did. This work is a lot more solid, although it's hard to summarize in a snappy way.

Everyone knows about the four bases of DNA (A, T, C, G). What this team has done is force bacteria to use a substitute for the T, thymine - 5-chlorouracil, which has a chlorine atom where thymine's methyl group is. From a med-chem perspective, that's a good switch. The two groups are about the same size, but they're different enough that the resulting compounds can have varying properties. And thymine is a good candidate for a swap, since it's not used in RNA, thus limiting the number of systems that have to change to accommodate the new base. (RNA, of course, uses uracil instead, the unsubstituted parent compound of both thymine and the 5-chloro derivative used here).

Over the years, chlorouracil has been studied in DNA for just that reason, and it's been found to make the proper base-pair hydrogen bonds, among other things. So incorporating it into living bacteria looks like an experiment in just the right spot - different enough to be a real challenge, but similar enough to be (probably) doable. People have taken a crack at similar experiments before, with mixed success. In the 1970s, mutant hamster cells were grown in the presence of the bromo analog, and apparently generated DNA which was strongly enriched with that unnatural base. But there were a number of other variables that complicated the experiment, and molecular biology techniques were in their infancy at the time. Then in 1992, a group tried replacing the thymine in E. coli with uracil, with multiple mutations that shut down the T-handling pathways. They got up to about 90% uracil in the DNA, but this stopped the bacteria from growing - they just seemed to be hanging on under those T-deprived conditions, but couldn't do much else. (In general, withholding thymine from bacterial cultures and other cells is a good way to kill them off).

This time, things were done in a more controlled manner. The feat was accomplished by good old evolutionary selection pressure, using an ingenious automated system. An E. coli strain was produced with several mutations in its thymine pathways to allow it to survive under near-thymine-starvation conditions. These bacteria were then grown in a chamber where their population density was being constantly measured (by turbidity). Every ten minutes a nutrient pulse went in: if the population density was above a set limit, the cells were given a fixed amount of chlorouracil solution to use. If the population had falled below a set level, the cells received a dose of thymine-containing solution to keep them alive. A key feature of the device was the use of two culture chambers, with the bacteria being periodically swapped from one to the other (which the first chamber undergoes sterilization with 5M sodium hydroxide!) That's to keep biofilm formation from giving the bacteria an escape route from the selection pressure, which is apparently just what they'll do, given the chance. One "culture machine" was set for a generation time of about two hours, and another for a 4-hour cycle (by cutting in half the nutrient amounts). This cycle selected for mutations that allowed the use of chlorouracil throughout the bacteria's biochemistry.

And that's what happened - the proportion of the chlorouracil solution that went in went up with time. The bacterial population had plenty of dramatic rises and dips, but the trend was clear. After 23 days, the experimenters cranked up the pressure - now the "rescue" solution was a lower concentration of thymine, mixed 1:1 with chlorouracil, and the other solution was a lower concentration of chlorouracil only. The proportion of the latter solution used still kept going up under these conditions as well. Both groups (the 2-hour cycle and the 4-hour cycle ones) were consuming only chlorouracil solution by the time the experiment went past 140 days or so.

Analysis of their DNA showed that it had incorporated about 90% chlorouracil in the place of thymine. The group identified a previously unknown pathway (U54 tRNA methyltransferase) that was bringing thymine back into the pathway, and disrupting this gene knocked the thymine content down to just above detection level (1.5%). Mass spec analysis of the DNA from these strains clearly showed the chlorouracil present in DNA fractions.

The resulting bacteria from each group, it turned out, could still grow on thymine, albeit with a lag time in their culture. If they were switched to thymine media and grown there, though, they could immediately make the transition back to growing on chlorouracil, which shows that their ability to do so was now coded in their genomes. (The re-thymined bacteria, by the way, could be assayed by mass spec as well for the disappearance of their chlorouracil).

These re-thymined bacteria were sequenced (since the chloruracil mutants wouldn't have matched up too well with sequencing technology!) and they showed over 1500 base substitutions. Interestingly, there were twice as many in the A-T to G-C direction as the opposite, which suggests that chlorouracil tends to mispair a bit with guanine. The four-hour-cycle strain had not only these sorts of base swaps, but also some whole chromosome rearrangements. As the authors put it, and boy are they right, "It would have been impossible to predict the genetic alterations underlying these adaptations from current biological knowledge. . ."

These bacteria are already way over to the side of all the life on Earth. But the next step would be to produce bacteria that have to live on chlorouracil and just ignore thymine. If that can be realized, the resulting organisms will be the first representatives of a new biology - no cellular life form has ever been discovered that completely switches out one of the DNA bases. These sorts of experiments open the door to organisms with expanded genetic codes, new and unnatural proteins and enzymes, and who knows what else besides. And they'll be essentially firewalled from all other living creatures.

Postscript: and yes, it's occurred to me as well that this sort of system would be a good way to evolve arsenate-using bacteria, if they do really exist. The problem (as it is with the current work) is getting truly phosphate-free media. But if you had such, and ran the experiment, I'd suggest isolating small samples along the way and starting them fresh in new apparatus, in order to keep the culture from living off the phosphate from previous generations. Trying to get rid of one organic molecule is hard enough; trying to clear out a whole element is a much harder proposition).

Comments (17) + TrackBacks (0) | Category: Biological News | Chemical Biology | Life As We (Don't) Know It


COMMENTS

1. GC on July 6, 2011 9:36 AM writes...

Damn, that's a hell of a clever experiment setup.

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2. Chemjobber on July 6, 2011 10:59 AM writes...

Reminds me of my favorite quote from the X-files:

"A 5th and 6th DNA nucleotide. A new base pair. Agent Scully, what you are looking at... it exists nowhere in nature. It would have to be, by definition... extraterrestrial."

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3. Anon on July 6, 2011 11:16 AM writes...

Not as interesting as the movie Splice or Species

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4. Pallas Renatus on July 6, 2011 11:32 AM writes...

This is quite possibly the most ingeniously simple (and *coolest*, by far) experiment setup I've heard of in years.

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5. Yggdrasil on July 6, 2011 12:48 PM writes...

Newscientist published a feature talking about a different approach to creating a new form of life. Instead of messing with DNA replication, George Church and colleagues are using their newly-developed, high-throughput genome engineering tools to mess with translation. Specifically, they are reassigning codons within the genetic code. They claim to be close to eliminating the least common stop codon from the genome, leaving open the possibility that this codon could be reassigned to something like an unnatural amino acid. Eventually, if enough of the codons were reassigned, such bacterium would be resistant to viruses (since normal DNA would not be read properly in these organisms). These engineered bacteria would be helpful for industrial applications as one would worry less about the possibility of a rogue phage taking down the entire production line.

Here's a link to the piece for those that are interested: http://www.newscientist.com/article/mg21028181.700-evolution-machine-genetic-engineering-on-fast-forward.html

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6. Tom on July 6, 2011 12:52 PM writes...

Could this kind of setup be used to tune microorganisms to greater efficiencies in other realms? Biofuel production comes to mind...

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7. Dr. Manhattan on July 6, 2011 12:58 PM writes...

"As the authors put it, and boy are they right, "It would have been impossible to predict the genetic alterations underlying these adaptations from current biological knowledge. . ."

Wait a minute- one is mixing up cause and effect here. These experiments are very, very interesting, but it is not possible to clearly tell (except in the cases directly involved in thymine metabolism) which additional mutations are required to grow on chlorouracil and which mutations are the result of chlorouracil's apparent mutagenic potential.

These experiments do underline the tremendous adaptability of life, and should serve as a lesson in humility for genetic engineers (and I am one of those, from time to time). Nicely done, indeed!!

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8. Algirdas on July 6, 2011 3:31 PM writes...

Tom (#5):

engineering enzymes and entire cascades in microorganisms for biofuel production is a very active research area. See for instance Nature biotechnology v.29 p.346; or Science v.329 p.559.

Whether the setup from the Angewandte paper could be used for such purposes is hard to say. Perhaps it can be adapted in some clever way. Note that for the Cl-U incorporation experiment, the phenotype that needs to be screened for is just growth; for biofuel production, you need to screen for production of certain class of compounds - much harder and labour intensive.

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9. Zach on July 6, 2011 8:55 PM writes...

@Algirdas

In other words, the first is a selection, the second a screen. I've tried to think of ways to turn the second into a selection for while now. Not sure if it's actually possible, but that could be a very powerful system, not just for biofuels, but for any biosynthetic need.

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10. Derek Lowe on July 6, 2011 9:50 PM writes...

Zach, you and me both. I've been thinking about that on and off for quite a while now, and I can't help but feel that there has to be a way to do it. Here's hoping an idea hits one of us!

Have you read David Liu's recent papers on this subject?

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11. gippgig on July 7, 2011 3:28 AM writes...

Some viruses substitute different bases, for example T4 uses glycosylated hydroxymethylcytosine instead of cytosine.
A lot of related work has been done (unfortunately I don't remember the references for much of this):
A strain of B. subtilis was evolved back around 1983 as I recall that used (externally supplied) fluorotryptophan instead of tryptophan.
Many amino acids have been added to the genetic code; the best example is a strain of E. coli that biosynthesizes p-aminophenylalanine & incorporates it at UAG codons (JACS 125 935).
A 5th & 6th base have been added to DNA & replicated successfully.
If you define life as nontrivial self-replication, a totally unnatural life form has already been created. It is an abstract mathematical pattern (cellular automaton) in the aptly named Game of Life (pentadecathlon.com/lifeNews/2010/05/oblique_life_spaceship_created.html). "Intelligent design" works!

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12. lynn on July 7, 2011 9:20 AM writes...

Zach and Derek - You'd have to make the biofuel (or whatever product) promote the growth of the organism. Maybe engineering a symbiotic relationship with another organism that metabolizes or recognizes the biofuel and produces a substance which the biofuel producer lacks (like an amino acid. Or - leave out the symbiont - and create a sensor/feedback "machine" that feeds the required amino acid [or other growth-rate-limiting essential nutrient] in proportional response to production of the biofuel. Mutations leading to higher biofuel production lead to faster growth [due to amino acid feeding]. Of course the biofuel producer would probably accrue mutations allowing it to more efficiently use the amino acid better. So start with a multiple auxotroph and limit each nutrient sequentially.... I think it's doable - and I'm sure the biofuel folks have been working on this....Fun to think about.

Also - Incorporation of bromodeoxyuracil into DNA leads to sensitivity to light (long UV, IIRC). I wonder about chlorouracil. I'd bet these new organisms were light sensitive.

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13. Zach on July 7, 2011 5:41 PM writes...

@Derek

I had not heard of him until now. Just glancing at his website, looks flippin' awesome. I'll definitely have to read some of his stuff, thanks for the tip.

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14. Michael on July 8, 2011 11:50 AM writes...

Ian Malcolm quotes:

"Oohh, Aahh, that's how all of this starts, but then later there's the running and screaming"

Malcolm: "But again, how do you know they're all female? Does someone go into the park and, uh... lift up the dinosaurs' skirts?"

(Wu): "No, we control their chromosomes. It's really not that difficult. It just takes an extra chromosome developed at the right hormonal stage to make them male. We simply deny them that. You're implying that a group composed entirely of female animals will... breed?"

Malcolm: "No, I'm simply saying that life, uh... finds a way."

Malcolm: "I'll tell you the problem with the scientific power that you're using here: it didn't require any discipline to attain it. You read what others had done and you took the next step. You didn't earn the knowledge for yourselves, so you don't take any responsibility... for it. You stood on the shoulders of geniuses to accomplish something as fast as you could and before you even knew what you had you patented it and packaged it and slapped it on a plastic lunchbox"


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15. Michael on July 8, 2011 11:55 AM writes...

Oh, one more...

Malcolm: " Yeah, but your scientists were so preoccupied with whether or not they could, they didn't stop to think if they should."

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16. AutoDidact on July 8, 2011 4:28 PM writes...

Wow, I mean wow. I can't imagine the number of ways a simple technique like this could be use. Kind of like the equivalent to pcr for directed evolution. You could force organisms into extremely metabolically stressful scenarios that mirror human disorders and see what they come up with over repeated generations. Drug discovery by Darwin anyone?

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17. Ryan K. on July 9, 2011 7:40 AM writes...

With regard to supposed Arsenic-based life:

Even though I used to work in one of the world's premiere Main Group labs, I would think that this is as obvious to everyone as it is to me...

Arsenates esters, such as those that would replace Phosphates in the Phosphate-Ribose/Deoxyribose backbone, are not at all hydrolytically stable. As-O bonds have much lower energy than P-O bonds, and have much less of a covalent character. I'm not sure of the exact half-lives, but Arsenates are not stable even in pH-neutral water. Essentially, any supposed As-containing DNA/RNA would fall apart quickly, thus limiting the size of the potential polymers.

Not to mention the redox-instability of Arsenates compared to Phosphates. As(V) likes to go to As(III) pretty quickly.

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