This Week’s Finds (Week 316)

Here on this This Week’s Finds I’ve been talking about the future and what it might hold. But any vision of the future that ignores biotechnology is radically incomplete. Just look at this week’s news! They’ve ‘hacked the genome’:

• Ed Yong, Hacking the genome with a MAGE and a CAGE, Discover, 14 July 2011.

Or maybe they’ve ‘hijacked the genetic code’:

• Nicholas Wade, Genetic code of E. coli is hijacked by biologists, New York Times, 14 July 2011.

What exactly have they done? These articles explain it quite well… but it’s so cool I can’t resist talking about it.

Basically, some scientists from Harvard and MIT have figured out how to go through the whole genome of a bacterium and change every occurrence of one codon to some other codon. It’s a bit like the ‘global search and replace’ feature of a word processor. You know: that trick where you can take a document and replace one word with another every place it appears.

To understand this better, it helps to know a tiny bit about the genetic code. You may know this stuff, but let’s quickly review.

DNA is a double-stranded helix bridged by pairs of bases, which come in 4 kinds:

adenine (A)
thymine (T)
cytosine (C)
guanine (G)

Because of how they’re shaped, A can only connect to T:

while C can only connect to G:

So, all the information in the DNA is contained in the list of bases down either side of the helix. You can think of it as a long string of ‘letters’, like this:

ATCATTCAGCTTATGC…

This long string consists of many sections, which are the instructions to make different proteins. In the first step of the protein manufacture process, a section of this string copied to a molecule called ‘messenger RNA’. In this stage, the T gets copied to uracil, or U. The other three base pairs stay the same.

Here’s some messenger RNA:


You’ll note that the bases come in groups of three. Each group is called a ‘codon’, because it serves as the code for a specific amino acid. A protein is built as a string of amino acids, which then curls up into a complicated shape.

Here’s how the genetic code works:

The three-letter names like Phe and Leu are abbreviations for amino acids: phenylalanine, leucine and so on.

While there are 43 = 64 codons, they code for only 20 amino acids. So, typically more than one codon codes for the same amino acid. If you look at the chart, you’ll see one exception is methionine, which is encoded only by AUG. AUG is also the ‘start codon’, which tells the cell where a protein starts. So, methionine shows up at the start of every protein, at least at first. It’s usually removed later in the protein manufacture process.

There are also three ‘stop codons’, which mark the end of a protein. They have cute names:

amber: UAG
ochre: UAA
opal: UGA

UAG was named after Harris Bernstein, whose last name means ‘amber’ in German. The other two names were just a way of continuing the joke.

And now we’re ready to understand how a team of scientists led by Farren J. Isaacs and George M. Church are ‘hacking the genome’. They’re going through the DNA of the common E. coli bacterium and replacing every instance of amber with opal!

This is a lot more work than the word processor analogy suggests. They need to break the DNA into lots of fragments, change amber to opal in these fragments, and put them back together again. Read Ed Young’s article for more.

So, they’re not actually done yet.

But when they’re done, they’ll have an E. coli bacterium with no amber codons, just opal. But it’ll act just the same as ever, since amber and opal are both stop codons.

That’s a lot of work for no visible effect. What’s the point?

The point is that they’ll have freed up the codon amber for other purposes! This will let them do various further tricks.

First, with some work, they could make amber code for a new, unnatural amino acid that’s not one of the usual 20. This sounds like a lot of work, since it requires tinkering with the cell’s mechanisms for translating codons into amino acids: specifically, its set of transfer RNA and synthetase molecules. But this has already been done! Back in 1990, Jennifer Normanly found a viable mutant strain of E. coli that ‘reads through’ the amber codon, not stopping the protein there as it should. People have taken advantage of this to create E. coli where amber codes for a new amino acid:

• Nina Mejlhede, Peter E. Nielsen, and Michael Ibba, Adding new meanings to the genetic code, Nature Biotechnology 19 (2001), 532-533.

But I guess getting an E. coli that’s completely free of amber codons would let us put amber codons only where we want them, getting better control of the situation.

Second, tweaking the genetic code this way could yield a strain of E. coli that’s unable to ‘breed’ with the normal kind. This could increase the safety of genetic engineering. Of course bacteria are asexual, so they don’t precisely ‘breed’. But they do something similar: they exchange genes with each other! Three of the most popular ways are:

conjugation: two bacteria come into contact and pass DNA from one to the other.

tranformation: a bacterium produces a loop of DNA called a plasmid, which floats around and then enters another bacterium.

transduction: a virus carries DNA from one bacterium to another.

Thanks to these tricks, drug resistance and other traits can hop from one species of bug to another. So, for the sake of safe experiments, it would be nice to have a strain of bacteria whose genetic code was so different from others that it couldn’t share DNA.

And third, a bacterium with a modified genetic code could be resistant to viruses! I hadn’t known it, but the biotech firm Genzyme was shut down for three months and lost millions of dollars when its bacteria were hit by a virus.

This third application reminds me of a really spooky story by Greg Egan, called “The Moat”. In it, a detective discovers evidence that some people have managed to alter their genetic code. The big worry is that they could then set loose a virus that would kill everyone in the world except them.

That’s a scary idea, and one that just became a bit more practical… though so far only for E. coli, not H. sapiens.

So, I’ve got some questions for the biologists out there.

A virus that attacks bacteria is called a bacteriophage—or affectionately, a ‘phage’. Here’s a picture of one:

Isn’t it cute?

Whoops—that wasn’t one of the questions. Here are my questions for biologists:

• To what extent are E. coli populations kept under control by phages, or perhaps somehow by other viruses?

• If we released a strain of virus-resistant E. coli into the wild, could it take over, thanks to this advantage?

• What could the effects be? For example, if the E. coli in my gut became virus-resistant, would their populations grow enough to make me notice?

and more generally:

• What are some of the coolest possible applications of this new MAGE/CAGE technology?

Also, on a more technical note:

• What did people actually do with that strain of E. coli that ‘reads through’ amber?

• How could such a strain be viable, anyway? Does it mostly avoid using the amber codon, or does it somehow survive having a lot of big proteins where a normal E. coli would have smaller ones?

Finally, I can’t resist mentioning something amazing I just read. I said that our body uses 20 amino acids, and that ‘opal’ serves a stop codon. But neither of these are the whole truth! Sometimes opal codes for a 21st amino acid, called selenocysteine. And this one is different from the rest. Most amino acids contain carbon, hydrogen, oxygen and nitrogen, and cysteine contains sulfur, but selenocysteine contains… you guessed it… selenium!

Selenium is right below sulfur on the periodic table, so it’s sort of similar. If you eat too much selenium, your breath starts smelling like garlic and your hair falls out. Horses have died from the stuff. But it’s also an essential trace element: you have about 15 milligrams in your body. We use it in various proteins, which are called… you guessed it… selenoproteins!

So, a few more questions:

• Do humans use selenoproteins containing selenocysteine?

• How does our body tell when opal is getting used to code for selenocysteine, and when it’s getting used as a stop codon?

• Are there any cool theories about how life evolved to use selenium, and how the opal codon got hijacked for this secondary purpose?

Finally, here’s the new paper that all the fuss is about. It’s not free, but you can read the abstract for free:

• Farren J. Isaacs, Peter A. Carr, Harris H. Wang, Marc J. Lajoie, Bram Sterling, Laurens Kraal, Andrew C. Tolonen, Tara A. Gianoulis, Daniel B. Goodman, Nikos B. Reppas, Christopher J. Emig, Duhee Bang, Samuel J. Hwang, Michael C. Jewett, Joseph M. Jacobson, and George M. Church, Precise manipulation of chromosomes in vivo enables genome-wide codon replacement, Science 333 (15 July 2011), 348-353.


Pessimists should be reminded that part of their pessimism is an inability to imagine the creative ideas of the futureBrian Eno

32 Responses to This Week’s Finds (Week 316)

  1. DavidTweed says:

    There’s another instance of genetic code modifying here. The abstract is opqaue to a non-specialist

    The ribosomal incorporation of nonnative amino acids into polypeptides in living cells provides the opportunity to endow therapeutic proteins with unique pharmacological properties. We report here the first clinical study of a biosynthetic protein produced using an expanded genetic code. Incorporation of p-acetylphenylalanine (pAcF) at distinct locations in human growth hormone (hGH) allowed site-specific conjugation with polyethylene glycol (PEG) to produce homogeneous hGH variants. A mono-PEGylated mutant hGH modified at residue 35 demonstrated favorable pharmacodynamic properties in GH-deficient rats. Clinical studies in GH-deficient adults demonstrated efficacy and safety comparable to native human growth hormone therapy but with increased potency and reduced injection frequency. This example illustrates the utility of nonnative amino acids to optimize protein therapeutics in an analogous fashion to the use of medicinal chemistry to optimize conventional natural products, low molecular weight drugs, and peptides.

    What this means is that they’ve made the protein more potent by increasing the time it takes to get filtered by the kidneys, which they’ve done by adding “unusual non-active stuff” that makes it heavier. (Very clear explanation from ars technica here.)

  2. This could increase the safety of genetic engineering.

    Which reminds me of a Japanese nuclear power plant which was made safer by building a tsunami wall around it…

    Anyhow I’m not really scared by genetic tinkering (enough harm is done in conventional ways, like feeding antibiotics to animals).

    I doubt biotechnology (in the above sense) will play any decisive role for the future. The future (if Homo S “Sapiens” is actually interested) would more be “ordinary” biotechnology, like non-destructive agriculture, forestry, ecosystems construction, etc.

    • John Huerta says:

      There’s enough of a chance that we could engineer some bacteria to do something very useful, such as processing cellulose into an unusually high yield of ethanol, that biotechnology remains worth pursuing. I bet you’ve seen this article before:

      http://news.cnet.com/2300-13840_3-6246653.html

      • I don’t think it’s not worth pursuing – I just think it won’t have an impact like e.g. the steam engine, or won’t be crucial in solving world hunger.

      • John Baez says:

        I believe biotechnology will have an enormous impact. We’re still just in the very early phases of groping around, but as people learn how to refashion life, we’ll see amazing things. Ultimately, if our civilization lasts long enough, biotechnology and nanotechnology will thoroughly blur the distinction between humans, other life forms, and technological artifacts.

        However, it may not solve world hunger, or global warming: those problems may become big too soon for this new technology to make a significant difference. It’s hard to tell.

        Here’s something from my 20 August 2005 diary entry:

        Out of the blue I feel like talking about the coming revolution in biotechnology. This will go much further than most people realize. Pretty soon, we won’t just be tinkering with the DNA of existing creatures, modifying it a little here or there. We’ll be creating completely new creatures and even new genetic codes.

        This is called "synthetic biology". The First International Meeting on Synthetic Biology was held in the summer of 2004, with talks like "Rewiring cell signaling pathways", "Programming cells and synthetic gene networks", and "Biological property rights".

        But in fact, Thomas Knight has been teaching classes on synthetic biology for several years – back at my old school, MIT. The kids in these classes use a toolkit of standardized parts called Biobricks to build new biological systems. It’s sort of like building electrical circuits from resistors, capacitors, and transistors. They do this for fun during the Independent Activities Period – a kind of free-for-all that takes place each January between semesters.

        Check out some of their projects! You’ll see stuff like:

        The objective of the project is to design a bacteria that when cultured will produce a recognisable polka dot pattern in the culturing medium. Our design attempts to achieve this by hijacking the quorum sensing mechanism employed by bacteria such as Vibrio fischeri and more particularly in our case Pseudomonas aeruginosa used to regulate group behaviour. We are attempting to use the las/rhl quorum sensing system used by the latter, in conjunction with a heat trigger to cause small clumps of bacteria to turn on the LacZ colour expression gene and hopefully get a small selection of polka dots in a tasteful display of purple.

        Some people will find this amusing. Some will find it exciting. Some will find it terrifying. I mainly just wish more people knew this kind of stuff is going on!

        • if our civilization lasts long enough, biotechnology and nanotechnology will thoroughly blur the distinction between humans, other life forms, and technological artifacts.

          If. And even if, it still sounds a bit science fiction to me. Sure there will be awesome wonders of “life” engineering, and like with Moore’s law progress in this art will accelerate. Apropos Moore’s law: Do we have thinking computers meanwhile?

          The point of my doubts is: Life consists not of isolated machines. It requires an ecosystem where energy (nutrients) and waste can be cycled in a sort of global metabolism. A Gaian view of sorts. As long as we don’t have a technological grasp on ecosystems (let alone the global one) bioengineered life will just flourish in artificial niches and have not much relevance to the greater picture.

          To reformulate my objection: I won’t bet the farm (literally) on biotechnology.

        • Hmmm. OK, here’s a challenge that would change my mind: Reconstruct and improve photosynthesis.

        • Frederik De Roo says:

          It has already been done – but not by humans ;-)

          C4

        • I could change my mind before breakfast. The art I’m waiting for might be named symbiotic engineering (in the sense of Lynn Margulis’ Symbiotic Planet). Paradigm: Enhanced chloroplasts.

          Give me C5…

        • reperiendi says:

          One thing I’d be interested in seeing is using fast, cheap sequencing to breed two closely-related species back into one, like horses and donkeys or South-American cats and African cats.

          Currently, horses and donkeys produce sterile mules, but one might imagine selecting horses that are more and more donkey-like and donkeys that are more and more horse-like, perhaps with some human editing of the genome, so that they eventually become able to breed.

        • John Baez says:

          Florifulgurator wrote:

          OK, here’s a challenge that would change my mind: Reconstruct and improve photosynthesis.

          A tiny step in that direction:

          • David Wendell, Jacob Todd and Carlo Montemagno, Artificial photosynthesis in ranaspumin-2 based foam, Nano Letters, 5 March 2010.

          It uses frog goop.

        • nad says:

          I also think that biotechnology will have a big impact. And it is indeed hard to tell, whether or how much it will help in overcoming world problems. One problem is also that people will first pick the (comparatively) “easy” challenges. That is it is way easier to modify (e.g. via trial and error) given genetic code, than to design a new one. And it is easier to design a “simple” entity (like a virus) than a complex one. Reverse engineering for complex organisms is still very hard. This is not only due to the sheer number of genes in a complex organism but in particular also how the clocks of gene expression work a.s.o.

          So for all sorts of reasons (e.g. alone for the reason that people in research may need to have a result for getting their next 3 month funding), there may be a lot of biotechological projects, where people tinker around without too much care for further implications. And tinkering around may be also detached from scientific contexts. In particular having such rather “simple” modifications at hand like the one linked to from here this may also raise the danger of bio-terrorism and -warfare. (Some people think that this could be worse than the threat from nuclear technologies and they may be right.)

          It is also a question in how far the patenting business etc. impairs the development of eventually useful technology. In particular high fees for licensing and information are a problem for developing countries and they are rather not a problem for terrorists.

          Optimists should be reminded that part of their optimism is an inability to imagine the creative ideas of the future – Brei Eno

        • Graham says:

          nad – I was thinking of modifying Brian Eno’s quote like that. But who is Brei?

  3. Jeremy Henty says:

    Ed *Yong*, not Young!

  4. Graham says:

    JB said:

    To what extent are E. coli populations kept under control by phages, or perhaps somehow by other viruses?

    The only figure I know relate to marine micro-organisms, which are by weight about 5% eukaryote, 90% bacteria, and 5% viruses. I think that means that viruses are a considerable problem for bacteria.

    If we released a strain of virus-resistant E. coli into the wild, could it take over, thanks to this advantage?

    I don’t see why not. An organism that multiplies every hour (which is what E Coli do in mammalian guts) only needs a tiny selective advantage to take over in a few years.

    What could the effects be? For example, if the E. coli in my gut became virus-resistant, would their populations grow enough to make me notice?

    You would certainly notice if a virus-resistant pathogenic strain took over. There are outbreaks of these pathogenic strains of course, like the recent one in Germany. As far as I know, the reason these kill tens rather than millions is that they are outcompeted by benign strains.

    What are some of the coolest possible applications of this new MAGE/CAGE technology?

    I think that is the wrong question because Craig Venter’s technology is so much more powerful.

    • Marc Harper says:

      Graham wrote:

      The only figure I know relate to marine micro-organisms, which are by weight about 5% eukaryote, 90% bacteria, and 5% viruses. I think that means that viruses are a considerable problem for bacteria.

      Note that since phages are so much less massive this implies that phages greatly outnumber bacteria in abundance. See the chart here for abundance comparison:

      Indeed, marine phages may be the most abundant DNA-based replicators on the planet and globally affect evolution and bacterial populations (see https://secure.wikimedia.org/wikipedia/en/wiki/Marine_bacteriophage for sources).

      I don’t see why not. An organism that multiplies every hour (which is what E Coli do in mammalian guts) only needs a tiny selective advantage to take over in a few years.

      Viruses evolve too, so there is no reason to expect a selective advantage to be sustainable in the long run. Of course this depends on the definition of “virus-resistant”. Since viruses use some of the same mechanisms to access a cell that cells use for other purposes, and with so many viruses out there, engineering a permanent resistance to viral infection would be a monumental feat. Whether this confers a selective advantage would depend on many factors including what is sacrificed in return for the resistance (if anything) and the resource requirements needed to maintain the resistance (if any).

      We do know that essentially all fruit flies contain a genetic sequence originally introduced by genetic engineering, so fixation of synthetic genetic changes is certainly possible. From https://secure.wikimedia.org/wikipedia/en/wiki/P_element :

      A P element is a transposon that is present specifically in the fruit fly Drosophila melanogaster and is used widely for mutagenesis and the creation of genetically modified flies used for genetic research. The P element gives rise to a phenotype known as hybrid dysgenesis.

      P elements were discovered in the 1970s when laboratory strains used since 1905 were compared to wild type flies. It seemed that these P elements had swept through all wild type populations of D. melanogaster subsequent to the isolation of the laboratory strains, which did not contain P elements.

      • Marc Harper says:

        On further review, I should clarify that P elements probably fixated within the last 100 years and arrived via horizontal gene transfer but not necessarily due to genetic engineering — the elements could have come from other species in the wild. See here for lots of information (http://engels.genetics.wisc.edu/Pelements/Pt.html).

        Nevertheless, the point is that non-vertically introduced genetic changes can fixate rather quickly.

    • John Baez says:

      Graham wrote:

      You would certainly notice if a virus-resistant pathogenic strain took over. There are outbreaks of these pathogenic strains of course, like the recent one in Germany. As far as I know, the reason these kill tens rather than millions is that they are outcompeted by benign strains.

      Thanks for all your comments.

      So, here’s a scenario that came to mind. A biotech company like Genzyme develops a version of E. coli with a modified genetic code, to make it virus-resistant. As Marc notes:

      … engineering a permanent resistance to viral infection would be a monumental feat.

      But maybe as long as the E. coli are kept safely contained in the laboratory, they’ll meet rather few challenges from viruses, so new strains of viruses able to hijack their genetic code won’t evolve.

      But then, a sophisticated terrorist group obtains a sample of these modified E. coli, and manages to make it more virulent, perhaps borrowing a virulence factor from E. coli O157:H7: the pathogenic strain that caused that outbreak in Germany (and elsewhere in Europe).

      So, this new strain of E. coli outcompetes the benign strains, spreads around the world, and kills millions…

      Of course it would take some sophistication to take an E. coli with a modified genetic code and give it genes from ordinary virulent E. coli: you might need to translate those genes into the new code. But maybe that wouldn’t be extremely hard, if the new code was public knowledge! I’m not sure, since I’m no expert.

      On the other hand, HR wrote:

      I don’t know the answer to how much bacteria are controlled by bacteriophages in the wild but laboratory strains of bacteria are already engineered with multiple metabolic mutations which are meant to make their persistence in the environment difficult.

      So, maybe we’re safe.

      By the way, apparently E. coli O157:H7 has genes that produce a Shiga-like toxin, which kills cells lining small blood vessels and produces bloody diarrhea. According to Wikipedia,

      Shiga-like toxins are iron-regulated toxins that catalytically inactivate 60S ribosomal subunits of eukaryotic cells, blocking mRNA translation and causing cell death. Shiga-like toxins are functionally identical to toxins produced by virulent Shigella species. Strains of E. coli that express shiga-like toxins gained this ability due to infection with a prophage containing the structural coding for the toxin, and nonproducing strains may become infected and produce shiga-like toxins after incubation with shiga toxin positive strains. The prophage responsible seems to have infected the strain’s ancestors fairly recently, as viral particles have been observed to replicate in the host if it is stressed in some way (e.g. antibiotics). The periplasmic catalase is encoded on the pO157 plasmid, and is believed to be involved in virulence by providing additional oxidative protection when infecting the host.

      I don’t know what all this means, but a ‘prophage’ is a virus that’s been integrated into the DNA of a bacterium.

      Graham wrote:

      John wrote:

      What are some of the coolest possible applications of this new MAGE/CAGE technology?

      I think that is the wrong question because Craig Venter’s technology is so much more powerful.

      Could you say a bit about that? I know Venter is doing interesting things, but is it doing something similar to this MAGE/CAGE stuff and doing it better, or is it doing something really different that’s just a whole lot more exciting?

      • Graham says:

        I am not an expert on this stuff, and worse, there doesn’t seem to be anybody reading this blog who is. But as I understand it:

        You could type any string of As,Cs,Gs,Ts, into your word processor and Venter’s technology would make an organism with that DNA. It is more complicated than printing your document, and I don’t think it is guaranteed to be entirely WYSIWYG, but that is the principle. It might take a few years and cost a few million, and of course it wouldn’t work unless you knew what to type, but it seems extremely flexible. As far as I can see the MAGE/CAGE stuff can only do limited edits. For example, one thing I think that could be done with Venter’s technology would be to remove the ‘junk’ DNA from a bacteria, making a more efficient version that could outcompete the naturally occurring version.

        • Both MAGE and whole genome assembly (WGA) can (in principle) make whatever genome that you want. In practice, this is more like writing software in which considerable testing and debugging is required the further you get away from a working design. So you need to make billions of combinations, check those that work, make some more, etc. This is very inexpensive with MAGE — 4.3 billion genomes per $1000 rather than $4 million per one genome. http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html

        • @ George Church Oct 9, 2011:

          George, thanks for the Nature reference. I’m involved in some re-assessment of simulated evolutionary methods, and some of the real-world techniques this article describes may have relevance. Also, while I’m at it:

          @ Graham July 21, 2011:

          Optimization is always a mixed blessing, one that in general gains improved performance within a narrow range of problems at the expense of generality and/or adaptability for some larger range of problems. Think race horses, but the examples are far broader and go far deeper than that. I’m convinced that one of the big tricks that we still don’t understand well in genetics and biology in general is some sort of rather splendid balancing act that looks at performance, generality, and adaptability as an interrelated suite of optimizations that works well not just individually, but in the overall composition and competition of many, many organizations, each with its own particular balance. E.g. look at fast mutation in RNA viruses versus stability in DNA viruses. Which is right? Neither, I suspect, at least not on any absolute scale. The question is whether each one does well at accessing a niche created by that overall interaction of many, many factors, including in particular other forms of life.

          There is a big opportunity in all of this for energy methods based on bio-modification, since as fast moving and sentient directors of molecular evolution we can access extreme optimizations that without special support would almost certainly be fatal competitively, and so do not occur naturally. That is something we should keep an eye out for when trying to use biology to solve the deeper problem of how to create a more virtuous energy capture and use cycle.

  5. Boris Borcic says:

    Hello there :) Ever since Azimuth started, I’ve been hesitating to participate, not being very clear on my own capacity to contribute better than noise. A few months ago I went as far as looking for a pseudo for registering, and that’s how I learned of the bacteriophage phi6 (which then distracted me). I am reminded of it by your question on phage regulation of E. Coli.

    Besides nicely evoking the name of physics, that bacteriophage phi6 should be most relevant to climate change among phages. That’s because it regulates the phytoparasitic pseudomonas syringae. P. syringae enrolls frost to damage plants, using proteins that raise the freezing points of water. According to wikipedia, it appears to play a significant role as an atmospheric condensation nucleator and the production of rain, snow, and hail.

  6. Alexander Vlasov says:

    Interesting question is Turing universality of similar “word processing” (splicing systems or so) operations with DNA. I remember, it was proven in some experiments about ten years ago.

  7. Blake Stacey says:

    This third application reminds me of a really spooky story by Greg Egan,

    Well, that doesn’t really narrow it down …

    called “The Moat”.

    Oh, OK. (-:

  8. HR says:

    On virus/bacteriophage

    The Genezyme drug is produced in eucaryotic cells (Chinese Hamster Ovary cells I think). These are not bacteria but mammalian cells. Bacteriophagse can contaminate bacterial cultures and kill them but they pose no threat to human. So while blocking bacteriophage activity makes manufacturing (and ultimately commercial) sense to the biotechs it’s not really a safety issue.

    Bacteriophages do exist in the wild, even in our guts. Scientists are researching the use of these beasts as treatment for bacterial infections. I think Soviet scientists first isolated these beasts from sewage and began using them many decades ago as an alternative to antibiotics. I work with a clinician/scientist in Manchester, UK who was trying to resurrect this treatment.

    I don’t know the answer to how much bacteria are controlled by bacteriophage in the wild but laboratory strains of bacteria are already engineered with multiple metabolic mutations which are meant to make their persistence in the environment difficult. These selection mutations are used to manipulate the bacteria in experiments allowing for selection of strains carry desired features but also have the added advantage of limiting growth in the wild. You can look at the phenotype of commonly used E. coli strains here:

    http://openwetware.org/wiki/E._coli_genotypes

  9. Terry Bollinger says:

    Regarding the importance to life of a wee sprinkling of selenium, don’t neglect mysterious molybdenum. While not part of any amino acids, Mo is fundamental to a surprisingly wide range of life-critical enzymes. Molybdenum is also quite rare. Best of all, it’s a whole lot harder to say five times fast in a row than is selenium.

    • John Baez says:

      Hi, Terry! Long time no see!

      Yes, molybdenum is perhaps the most bizarre of the elements required for human life. But some bacteria use tungsten, and strangely enough, sometimes all these weirder elements get used in conjunction:

      Tungsten, at atomic number 74, is the heaviest element known to be biologically functional, with the next heaviest being iodine (Z = 53). Although not in eukaryotes, tungsten is used by some bacteria. For example, enzymes called oxidoreductases use tungsten similarly to molybdenum by using it in a tungsten-pterin complex with molybdopterin (molybdopterin, despite its name, does not contain molybdenum, but may complex with either molybdenum or tungsten in use by living organisms). Tungsten-using enzymes typically reduce carboxylic acids to aldehydes. However, the tungsten oxidoreductases may also catalyse oxidations. The first tungsten-requiring enzyme to be discovered also requires selenium, and in this case the tungsten-selenium pair may function analogously to the molybdenum-sulfur pairing of some molybdenum cofactor-requiring enzymes. One of the enzymes in the oxidoreductase family which sometimes employ tungsten (bacterial formate dehydrogenase H) is known to use a selenium-molybdenum version of molybdopterin. Although a tungsten-containing xanthine dehydrogenase from bacteria has been found to contain tungsten-molydopterin and also non-protein bound selenium, a tungsten-selenium molybdopterin complex has not been definitively described.

      I’m pretty bummed that nobody has stepped forward to answer my questions about selenocysteine. Obviously there aren’t enough biologists reading this blog! I may have to do a little library research to satisfy my curiosity.

  10. Terry Bollinger says:

    Tungsten! That’s about as weird as uranium, and it’s a biological element about which I’ve never seen anything before. Surely this unexpected dependence makes Stephen Wolfram feel more needed, at least by European bacteria?

    While not advocating it — I’m a big fan of sometimes just saying “there is insufficient data to formulate a testable theory” — I would of note that close associations of odd elements are the meat of panspermia speculations.

    The odd element combinations, if primordial, would from that perspective tend to indicate that the true origins of life were on planets very different indeed from those of our solar system. Such a point could in turn help explain why speculations about the origin of life on earth always seem to end up stretched to some statistical limit. If life originated in a very different setting, theories of how life might have originated on earth would be roughly like trying to explain the presence of diamonds in a creek bed by theorizing how room-temperature aqueous processes could lead to diamond formation in streams.

    Conversely, perhaps on some very distant and very heavy planet, the chemical quirks and coincidences that baffle us about life might look far more mundane and commonplace.

    It’s worth noting also that such speculations could perhaps someday lead to computational testability of panspermia ideas.

    More specifically: If one modeled in detail the surface chemistry of an energetic planet rich in tungsten, molybdenum, selenium, and other plausibly associated elements, would that model show a large increase in the likelihood of physicochemical processes that exhibit expandable memory, replication, and mutation?

    As with computational astrophysics, such an approach might take panspermia from the realms of interesting but basically idle speculation into something for which non-trivial results might be derived about the type of extrasolar environment in which life most likely arose.

    I must at the same time admit that I don’t think any currently existing computational and modeling systems exist that could handle the massive simulations that would be needed.

    —-

    Did I just say “theories of aqueous diamond formation? Hmm!

    What follows is a total tangent, something I worked on back in high school, which was… a while back!:

    Why does life not create diamond for things such as teeth?

    Before you laugh too quickly about the need for intense heat and pressure or vacuum vapor deposition, beware: Those are all sledge hammer approaches, methods that encourage a tiny and energetically minimal change in bonding direction by slamming carbon around in ways that give it little choice.

    Life encourages tiny changes in bond directions all the time, and does so elegantly and with minimal fuss. The tools of course are called enzymes. As you read this page, said enzymes are constantly creating bonds whose statistical unlikelihood dwarfs by many orders of magnitude the difference between graphite and diamond.

    So again the question, but a bit differently: Why does life not create the enzymes to create diamond for uses such as teeth?

    Energy levels are not the issue. The difference between those of graphite and diamond are well within the limits that life can handle. (Please note however that I am going on the memory of calculations I made decades ago!)

    Size? Maybe. Graphite and diamond bonds are very compact. It could simply be that the types of protein-based enzymes made by life simply can’t handle the smaller bond sizes. I don’t fully buy that argument, however, since life long ago learned the trick of using big tools to make smaller tools. Thus even if a first-order DNA-defined protein enzyme cannot do the trick, it and other enzymes could very likely create smaller chemical molecules — catalysts — with just the right bonding structures.

    Ignorance? It’s possible! It took life a couple of billion years to reach the specific set of higher-efficiency photosynthesis seen in grasses, so who’s to say that life just hasn’t been around long enough to figure out how to make diamond?

    And with that “hmm, what if…?” thought, I’ll end with my aqueous diamond synthesis model from back in the late 1970s.

    First, cut a seed diamond to expose its cubic faces. Next, treat the surface with hydrogen to ensure termination of carbon surface bonds with hydrogen atoms.

    What this gives you geometrically is a surface lined with alternating close pairs of hydrogen atoms that are first vertical (:) and then horizontal (..), like this: (: .. : .. : .. :), only with the horizontal dot pairs better centered. At high magnification the diamond surface would look a bit like one of those metal traction surfaces that use alternating vertical and horizontal short bars, only with dot pairs replacing the bars.

    Now immerse the surface treated in water and add your reactant and catalyst. The reactant is formaldehyde, and the catalyst binds to the formaldehyde so that the oxygen end sticks out.

    The loaded catalyst has additional groups that bond selectively to the hydrogen-terminated cubic diamond surface. As a loaded catalyst molecule approaches the cubic diamond surface, it presents the oxygen to one of the hydrogen pairs at just the right angle to cause the production and discarding a water molecule:

    OCH2 [formaldehyde] -> C [graphite or diamond] + H2O

    Again from memory, I’m pretty sure that this reaction is energetically favorable, so no additional energy should be needed. Even if an energy boost was required, though, it could be provided by a more complex catalyst system. That is after all what the ATP cycle in cells is all about.

    The result: A new layer of diamond begins to form. The nice thing about using the cubic faces (versus the octahedral ones) is that geometry alone determines correct bonding. As long as the formaldehyde oxygen atoms bond only to the close pairs of hydrogen atoms, the resulting layer is geometrically guaranteed to have diamond bonding, versus graphite or other allotropes.

    Layer-by-layer growth should be possible using only a single catalyst. However, adding a second edge-growth catalyst would likely speed up the process. This second catalyst would cause the formaldehyde to bond in exactly the same fashion, but would attach itself at the growing edge of a new diamond layer.

    The oddest thing about all of the above is that I’ve never been able to come up with a powerful reason why it could not work.

    The configuration is simple, the energy levels are not drastic, and real enzymes do much more complex things than this. Back when I first worked on this idea would have liked to model the detailed bonding behavior of the formaldehyde oxygen as it approaches the diamond cubic surface hydrogen pair. But alas, I didn’t get my first four function hand calculator until my senior year in high school, and I don’t think the quantum chemistry models of that time would have been up to the task anyway.

    So: Could diamonds be grown in aqueous solutions?

    Maybe; maybe not. I would at least suggest that it is a question that merits more careful examination. I don’t think it can be fully dismissed based on analogies (pressure and vacuum methods) that don’t directly address the question of enzymatic plausibility.

  11. Bill says:

    As a mathematics major turned biologist, I have been lurking here for a while. My topology is really rusty since it has been >30 yrs since I took the course, along with basic algebra, as an undergraduate. I have been interested in what you have been talking about and have some ideas. But that will come later. First, some answers to your questions.

    • What did people actually do with that strain of E. coli that ‘reads through’ amber?

    Nonsense mutations and suppressors. Nonsense mutations have a change of a codon for an amino acid to a stop codon (TAA, TGA, TAG). These were among the last codons to be described (mid 60′s). The amber codon was discovered through its suppressor mutation, a change in the anticodon of a tRNA that allowed it to recognize the amber codon (UAG). These suppressor strains were an essential component of the microbial geneticist’s tool kit for quite a while, especially for bacteriophage geneticists. Isolating an amber mutation in a bacteriophage in your gene of interest allowed you to grow the phage and then infect a wild type strain. Without the presence of the suppressor, the mutant phage would no longer express that gene and one could then do experiments to determine the gene’s function. Such a mutation is referred to as a conditional lethal. Another widely used conditional lethal are temperature sensitive mutants (ts).

    Many, many more conditional lethals have been developed for many different systems, and these have been invaluable to microbial geneticists (and here I include yeast genetics). The basic game played is one that takes + –> – and then – –> +. For example, say you have isolated a mutation that affects a certain process. What other proteins are involved in the process? One way of isolating mutations in other genes is to find extragenic suppressor mutations that restore function (- –> +). With new genes, the whole game can be played again to define the network of genes for a given process.

    When this sort of technology is combined with biochemistry, it provides a very powerful tool for studying fundamental biological processes. It’s no wonder that E. coli (Gram negative), B. subtilis (Gram positive), S. cerevisiae (baker’s/brewer’s yeast) and S. pombe (fission yeast) have been the model organisms of choice.

    • How could such a strain be viable, anyway?

    For amber mutations, the efficiency of suppression is ~30 % for the good suppressors. Also, there are only ~300 amber stop codons in E. coli. Thus, relatively few proteins will be affected. Moreover, a second stop codon in the same reading frame will soon terminate translation leading to a normal protein with some length of junk on the end. Their are several different fates.

    First, the extra sequence could have no or little effect on protein function. Indeed, this is often the case, and biologists have taken advantage of this to create genes encoding protein-protein hybrids. One type of fusion has changed the face of cell biology: GFP fusion. Here, the gene encoding GFP (green fluorescent protein) is placed in the same reading frame downstream of your gene of interest with its stop codon removed, creating a C-terminal fusion. Similarly, GFP can be placed upstream, creating an N-terminal fusion. When expressed in a cell, the location of the GFP (and presumably the fusion) can be followed in real time in live cells. This is only one use of GFP (and the rainbow of color variants) and new uses are invented all the time.

    Second, the extra protein sequence could have a detrimental effect on the protein’s function. However, since 70% of the time translation is terminated normally, there is enough good protein to provide the needed function for the cell. This is analogous to recessive mutations, but in this case the mutant protein and the wild type are expressed from the same gene.

    Third, the extra sequence could prevent the protein from folding properly. This is usually not a problem for E. coli, or any other organism, since it contains systems that deal with unfolded or improperly folded proteins. First, chaperon proteins attach and try to refold the protein, but if this is unsuccessful, the protein will be taken to a conserved molecular machine that digests the protein back to amino acids, the molecular equivalent to a paper shredder. Even if this system is overwhelmed by over production of a foreign protein, E. coli effectively segregates the protein into what are called ‘inclusion bodies’. We take advantage of this for protein purification purposes since inclusion bodies are easy to isolate. The hard part is getting the protein back into solution and folded properly.

    • What are some of the coolest possible applications of this new MAGE/CAGE technology?

    The goals of Farren Isaacs & George Church and Jennifer Normanly & Jeffrey Miller are related. Normanly and Miller’s goal was motivated back in the days when genetic engineering was complicated and tedious. Essentially, their thought was that if one wanted to isolate mutations in a protein, you were always limited to those mutations that you could identify in your selection and screening procedure. However, one could isolate a collection of amber mutants across the gene and then use different amber suppressor strains that substituted a different amino acid into the site. The ideal situation would have been to be able to substitute each of the 19 other amino acids. Unfortunately, nature provided several roadblocks. One I know of is charging of amino acids to their cognate tRNAs by the synthetases. Some of them use the anticodon to select the proper tRNA for charging; change the anticodon and you get mischarging or little charging at all. When the project reached this point, the paper that you cite, PCR and other techniques essentially made mutagenesis easy, so the Normanly & Miller technique was not widely used. Good idea at the time.

    The goals of Isaacs & Church are more far reaching. Elimination of all of the amber stops will allow, as you surmised, the placement of that codon anywhere within a gene. In other related work, using frame shifting tRNAs, people have started to develop amino acyl-tRNA synthetases that will charge non-native amino acids onto tRNAs. It is hoped that this can expand the catalytic range of enzymes, plus a whole bunch of other applications (novel peptide drugs comes to mind). The disadvantage of the frame shifting approach is that it is inefficient and if one made it efficient it would probably kill the cell. In contrast, the Isaacs & Church approach avoids this difficulty in two ways. Once all of the used amber stop codons are eliminated, you can then safely remove the protein factor that recognizes UAG. There are two translation termination factors in E. coli RF-1 and RF-2. The these proteins that assume the shape of a tRNA and bind to the stop codon in the A-site of the translating ribosome. One RF recognizes UAA and UGA and the other UAA and UAG. Eliminating the latter will free up UAG for aa-tRNA binding and eliminate termination at UAG. Thus, 100% efficiency of inserting the desired amino acid into a site is at least theoretically possible. But as with everything in biology, who knows what can go wrong.

    • What could the effects be? For example, if the E. coli in my gut became virus-resistant, would their populations grow enough to make me notice?
    • If we released a strain of virus-resistant E. coli into the wild, could it take over, thanks to this advantage?

    It is unlikely that these sorts of modifications could make E. coli virus resistant. It’s analogous to the LHC creating a black hole that will gobble the earth up. Bacteria and viruses have been around for 4 billion years, and thus far, no virus resistant bacteria have ever been discovered or have taken over an ecosystem. Bacteria can become resistant to a particular strains of phage, but never to all phage.

    Bacteria do possess several defense mechanisms to inhibit incoming phage. The best studied is the restriction/modification system. This is a two gene system. One gene encodes a site specific DNAase that specifically cuts DNA. Discovery of these enzymes led to the first wave of genetic engineering of the cut and paste variety. The second gene encodes a DNA modifying enzyme that adds a methyl group to a base in the recognition site of the restriction enzyme. Different strains of bacteria have different restriction/modification systems. Thus, an incoming virus that grew on a cell with one system will not have the new host’s modification and thus will be digested. However, ~1/1000 incoming phage do survive (it’s a kinetic race between modification and digestion) and the progeny from that infection will have the new modification but not the old one. There is a plethora of defenses that phage have come up with to deal with this.

    A second, defense mechanism is the CRSPR system. It is really interesting but I have not read up on it, so I won’t comment.

    Finally, E. coli is a wimp. It is a minor member of the trillions of bacteria that live in your gut. Mouse studies have shown that it inhabits the mucous lining the intestine near the cecum. It normally grows there and nowhere else. It uses mucous polysaccharides as an energy/carbon source and uses the Entner-Doudoroff pathway for glycolysis (not the Embden-Meyerhof-Parnas pathway that everyone learns in into-Bio). Mutations in the ED pathway prevent E. coli from colonizing a host, and once it is shed from the mucous layer, it no longer grows. So even if you could get a virus resistant E. coli, nothing much would happen.

    • To what extent are E. coli populations kept under control by phages, or perhaps somehow by other viruses [Nomeclature note: bacterial virus === bacteriophage === phage]?

    The roles of phage in bacterial ecology are largely unknown. It was only about 10 yrs ago that the number of phage present in the environment was first uncovered. The numbers still addle my brain when I stop to think about it. Take any surface water and you can find 10^5 – 10^7 phage/ml. A popular estimate for the number of phage in the oceans is ~10^31. For bacteria that are rapidly growing, I have seen estimates anywhere from 20 to 50% of the bacteria are killed off each day. When a phage kills a bacterium, it causes the cell to burst and release the newly formed phage. This also releases nutrients for other bacteria to consume, which are then eaten by the zooplanktonic grazers and on up the food chain.

    It’s a no brainer to assume that bacteriophage are important players in carbon and other nutrient cycling. For example, cyanobacteria blooms are measured by satellite and used in calculation of the uptake of CO2 into the ocean. I haven’t delved into the assumptions surrounding CO2 flux into and out of the ocean, and in one talk I heard, no one in the field has a good handle on it. Experiments are just beginning.

    A second role, is one of gene transfer. While lytic phage just infect grow and kill their hosts, lysogenic phage, can grow in this manner but are also capable becoming quiescent, either integrating their DNA into the bacterial chromosome or by becoming a stable plasmid. Lytic gene expression is turned off by a repressor protein that binds to the lytic gene promoter regions. Some phage carry genes that are expressed in the lysogen. These can be beneficial, such as genes for photosynthesis and nitrogen fixation or harmful (from our perspective). In many of the pathogenic E. coli, the toxin genes were brought in by a lysogenic phage.

    There’s a separate story on how the study of phage led to the development of MAGE/CAGE technology, but I have written too much.

    • John Baez says:

      Wow, what a great reply! I’m glad my pleas led you to delurk.

      I have a few questions. At first this passage stumped me:

      The amber codon was discovered through its suppressor mutation, a change in the anticodon of a tRNA that allowed it to recognize the amber codon (UAG).

      But maybe now I get it. The modified tRNA was able to recognize the amber codon and create an amino acid from it, thus ‘suppressing’ its actual function as a stop codon?

      (At first I didn’t get how ‘recognizing’ counted as ‘suppressing’.)

      For amber mutations, the efficiency of suppression is ~30% for the good suppressors.

      So you’re saying that at most 30% of the amber codons get ‘read through’ and translated into an amino acid, while the rest still act as stop codons.

      However, ~1/1000 incoming phage do survive (it’s a kinetic race between modification and digestion) and the progeny from that infection will have the new modification but not the old one.

      Does “have the new modification” mean “have a methyl group attached to a base in the recognition site of the restriction enzyme”? I’m a bit confused because I don’t understand whether this modification in the phage’s DNA is something a bacterium does to help kill the phage, or something a phage needs to have to keep from being killed. Your passage above makes it sound like the progeny have this modification yet survive.

      On a vastly lesser note, I was surprised to discover that ‘phage’ is used as a plural noun as well as singular. It’s also true for ‘sheep’ and ‘fish’, but those are old Anglo-Saxon words for creatures that come in herds (or schools), so they function as mass nouns like `mud’ or ‘water’: we might say “a lot of fish swimming in a lot of water”. I suppose there are so many phages that they deserve to be treated as a mass noun, but it must have taken someone bold (or uneducated) to be the first to try that with a Greek-derived word!

      I have been interested in what you have been talking about and have some ideas. But that will come later.

      Okay, great. I’m looking forward to it.

  12. [...] A second comment is related to the Manhattan project itself but also to the dangers of biotechnology. [...]

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