The Newest Bacterium: Part 3, Finalé

6 Jul

How did the Venter research group synthesize a genome for transplant? 

The first thing I really wanted to know in reading the original research article in Science (May 20th, 2010) was, how did the authors of this ground-breaking piece of science manage to synthesize an entire bacterial genome? 

And the answer is: they didn’t, not exactly. 

If you’d like to see for yourself, the actual materials and methods used in the Science article were not contained within the article itself.  They are, however, available here as “Supplementary Materials.” 

But if you’d rather not sit down with your molecular microbiology text book and dictionary to go through their 29 pages of methods, let me explain as briefly as possible…

As I mentioned in the previous post on this (Part 2), Venter’s group of scientists at the J. Craig Venter Institute (JCVI) had already worked with these Mycoplasmas for quite some time and had sequenced at least one entire genome.  In doing so, as well as a series of experiments in which they could delete one gene at a time and see what happened to the bacterium, they managed to figure out precisely which genes were necessary for survival in the lab and which were not.

At that point they simply ordered the genome sequence, in little pieces, from a company called Blue Heron  that commercially manufactures DNA in the form of oligonucleotides (a.k.a. oligos); an oligo is a small piece of DNA arranged according to your (consumer) specifications. 

There are a variety of companies in existence all over the world that will put together oligonucleotides for any researcher or institution; you only have to tell the company what order you want the bases in (A, T, C, and G).  Venter and the scientists at the JCVI simply ordered the known sequence of the genome of M. mycoides from Blue Heron in pieces each about 1,080 bases long, and made sure that the pieces all had overlapping, matching sequence at the ends. 

So, the Venter group also wanted to make sure that their soon-to-be creation would not only function properly, but could be identified, patented, and its movement tracked.  Therefore, in writing this synthetic genome, they included some “watermark” non-coding sequences.  These are bits of genetic material that don’t actually get used in the daily life-functions of the bacterium; they are just there, taking up space, allowing recognition and quality assurance by the scientists working with the organism.  In these watermark sequences the scientists spelled out their own names, a few choice quotes, and even a webpage, all in the genetic code of this organism (more info on the code at another interesting blog).

But how did they assemble the pieces of DNA into a genome?

Two words:   slave   labor 

Only, not the way you might be thinking.  They used a common yeast (Saccharomyces cerevisiae; a.k.a. “bakers’ yeast” or “brewers’ yeast”) to put all the pieces together for them.  This particular yeast was the first fully sequenced eukaryotic (eukaryote = cells with a true nucleus, i.e. plants, animals, us, not bacteria) organism and is particularly easy to grow. 

Since its genome was sequenced more than ten years ago and it’s the “model organism” for eukaryotic cell function, we know a lot about it, how it works, but more specifically, how it’s DNA repair and synthesis mechanisms function. 

Dr. Venter and his team of scientists made use of this knowledge in the way they designed the pieces of the bacterial genome that they were interested in transplanting.  There were specific recognition sites allowing the natural machinery of the yeast cell to stitch them all together in a specific order.  They had to go through three rounds of yeast-facilitated patch-work-quilting to get the whole genome, starting with the small pieces they ordered from Blue Heron, then gradually making larger and larger pieces until one of their yeasts finally contained the entire “synthetic” Mycoplasma mycoides genome (plus watermark sequences).

My 2¢ on the whole thing…

The researchers at JCVI actually published another paper in Nucleic Acids Research only about 3 months earlier describing this entire process, entitled “Cloning whole bacterial genomes in yeast.”  They do a nice job of explaining that microbial gene cloning  has been done for years in microbiology laboratories all over the world for a variety of reasons.  Now, just read that last sentence again slowly and let it really sink in. Then think about this: In 1973, Stanley Cohen and Herbert Boyer actually created the first recombinant DNA (cloned) organism and we’ve been using this technology ever since. (Cloning technology development timeline)

This paper in Science which has caused all this hubbub simply pushed a common method a bit further, from just one gene or a few genes, into the ability to manipulate a very small bacterial genome.  One major driver to this type of research (amongst others) has been the difficulty in not being able to grow most microbes in the lab for detailed study. (The uncultured microbial majority)

If you can’t grow it in the lab, you know fairly little about the organism or what it does out in nature.  We can sequence the same microbe, but still we aren’t certain how that genetic code amounts to functionality in the environment.  The primary advantage to the JCVI’s newest technique over the cloning that we’ve been doing previously is that we can use much larger pieces of DNA, longer gene sequences, and this means more information

The limitations of this technique are obvious when we consider the fact that the transplanted genome was in fact that of Mycoplasma mycoides (with a few additions and deletions).  Not only was the genome specific to that organism, but all the constraints to making this experiment work will be equally as specific.  Try a different genus and the whole ballgame has changed. 

That said, I do acknowledge the fact that these two organisms and the M. mycoides genome may serve as a useful platform or starting place.  I would imagine next steps will be to put together sets of genes from a host of different organisms to create hybrids with the M. mycoides sequence, or some other very simplified genome. In this case, the donors of the genes tacked on to the base genome wouldn’t have to be limited to microbial species,  many genes and gene fragments have already been patented  amid some more recent controversy (roughly 20% of the human genome, in fact).  Each new hybrid, of course, would be patentable in its own right and you can easily see how lucrative this line of research will soon be.

Did I say I’d be brief?

So, in short, the research group at JCVI combined several techniques commonly used in the various fields of microbiology and transplanted the piecemeal genome of one organism into a closely related organism where it began to function rather normally as the first organism.  An interesting and noteworthy piece of science, it is not, in my opinion, the “creation of life” that merits the fear and awe with which the world reacted (at least not yet).

___________________________________________________________________________________________________________________________________________________________________________

 Gibson, D.G. et al. (2010) Creation of a bacterial cell controlled by a chemically synthesized genome.  Science, 329 (5987): 52-56.  DOI: 10.1126/science.1190719

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2 Responses to “The Newest Bacterium: Part 3, Finalé”

  1. microbialmodus July 12, 2010 at 12:42 pm #

    View “Comments on the ‘synthetic cell'” on July 8th via Small Things Considered… http://schaechter.asmblog.org/schaechter/2010/07/comments-on-the-synthetic-cell.html

  2. katesisco May 2, 2012 at 3:53 pm #

    Exactly what you would expect from a market-oriented, capitalist-controlled economy.

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