Making Microbes: Technical Milestones for Synthetic Genomics

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“For a chemist, poliovirus is nothing more than a chemical”

[C332,652H492,388N98,245O131,196P7,501S2,340 to be exact.]

It’s been one year short of a decade since Eckard Wimmer and colleagues from Stony Brook University in New York conjured up a self-replicating poliovirus from off-the-shelf laboratory chemicals, proving to the world that a “living” creature could be re-created by scientists without a natural template.  They showed that when atoms are put in the right order a particle emerges with all the properties needed to replicate and survive in nature. In other words, says Wimmer, when the synthetic genome enters a cell it is “booted” to become a real living poliovirus, hijacking what it needs from its host to reproduce and evolve. “This fascinating dual nature of viruses as nonliving and living entities –chemicals with a life cycle—has largely been ignored,” he adds.

But poliovirus is a particularly nasty sort of creature. When it gets into a human, it replicates in the gastrointestinal tract and from there can migrate into the central nervous system (CNS) where it targets the neurons that control muscle movement. Invasion of the CNS by poliovirus can result in irreversible paralysis and sometimes death.  So why would anyone want to cook them up in a laboratory?  Wimmer’s collaborator, Steffen Mueller, also at Stony Brook, explains that chemically synthesizing novel polioviruses with greatly debilitated CNS reproduction offers new ways to combat human disease.  More specifically, Wimmer believes that recoding viral genomes via “synthetic attenuated virus engineering (or SAVE), is a fast route to vaccine discovery.”  In fact, vaccine development is a major research objective of the Wimmer Lab scientists.

The common goal of all our work, says Wimmer, “is to better understand an organism’s properties, particularly its pathogenic armory.” But he also notes that their synthesized poliovirus is nearly identical to the wild-type virus and is thus not a product of synthetic genomics which “strives to generate biological systems that don’t exist in nature.”

RNA versus DNA viruses

RNA viruses such as polioviruses are sequenced by converting the genome

Replicating synthetic bacteria. Image Credit: J. Craig Venter Institute

into double-stranded DNA using a retroviral enzyme called reverse transcriptase. This technology has enabled whole-genome synthesis and generation of several other important RNA viruses including the 1918 ‘Spanish’ influenza virus which wasn’t isolated at the time of the outbreak and had to be reconstructed from scratch by Jeffrey Taubenberger and colleagues at NIH’s NIAID in Bethesda, Maryland ;  the Chimpanzee Immunodeficiency Virus (SIVcpz) which jumped species and was  responsible for the human HIV-1 pandemic;  and an infectious bat SARS-like coronavirus, the cause of a recent brief SARS pandemic.

Synthesizing DNA viruses is quite another matter altogether and only one, ΦX17, has been so far assembled. Hamilton Smith and colleagues working in J Craig Venter’s lab began with an already deciphered DNA sequence that they assembled in a test tube in two weeks and, in 2003, finally activated the resulting ΦX17 DNA into making live virions in the workhorse bacterium Escherichia coli. “The E. coli cellular machinery read the synthetic genetic DNA and produced the viral proteins, which self assembled to form the active virus,” according to Venter.

However, Wimmer says that ΦX17 is also a copy of an existing virus and, technically speaking, not a product of true synthetic genomics.

Colonies of the transformed M. mycoides bacterium. Image Credit: J. Craig Venter Institute

The first synthetic bacterium goes live

The synthesis of ΦX174 was only a test run for the Venter group’s assembly of the 582,970-bp bacterial genome in Mycoplasma genitalium.  In the 1990s, Venter, Smith and Clyde Hutchison III at the J. Craig Venter Institute in Rockville, Maryland and San Diego, California sequenced its DNA. That genome was synthesized but for unknown reasons could not be booted to life. It took a dedicated team of 20 people, an estimated $40 million and a different, much larger synthesized Mycoplasma genome (1,089,202 bp) to finally boot an artificial bacterium to life, a great feat announced by the Venter group with considerable fanfare on May 20th of last year. The entirely artificial M. mycoides genome, now in a bacterium known affectionately as “JCVI-syn 1.0,” was  constructed from four bottles of chemicals that make up DNA and was inserted into a bacterial cadaver. The resulting replicating bacterium is the first cell controlled completely by a synthetic genome.

But this still isn’t synthetic biology say some  because the JCVI team copied an existing plan. Nonetheless, the science that was developed to get this far is helping scientists engineer microbes that decontaminate toxic waste, track down tumors, secrete biofuels and medications as well as improve clean water technology.

Research details and more information can be found in:

Wimmer E, Mueller S, Tumpey TM, Taubenberger JK.  Synthetic viruses: a new opportunity to understand and prevent viral disease in Nature Biotechnology (2009); 27 (12), pp. 1163-1172.

Gibson DG, Glass JI, Lartigue C, et al. Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome in Science Express 20 May 2010; pp. 1-7.

What’s in a name? News Feature in Nature Biotechnology (2009); 27 (12), pp. 1071-1073.

Read more about:

Made-To-Measure Microbes here.

Genetic engineering here.

Synthetic biology here.

The Venter Group’s triumph here.

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