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Cell Biology, Genetics, Video

Article Review: Synthetic Genomics

by S. Lee Ware and Krystina Sandefur

Introduction

In “Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome”, Venter et al. report the first successful design, synthesis, assembly, and transplantation of an artificial genome into a host cell. The bacterial host subsequently underwent phenotypic transformation, resulting in the first engineered species created from a synthetic genome.

The first complete genome sequence was completed in 1977. In the decades since, the rate of digitization of genomic information has increased by 8 orders of magnitude, but, our understanding of genomics has not increased so rapidly; for example, no single organism’s genome is completely understood in terms of the biological function of each gene. This experiment sought to expand our limited genomic understanding by answering two questions: do chromosomes contain the entire genetic repertoire? And if so, can a complete genetic system be reproduced through chemical synthesis, starting with digitized DNA?

The JCVI team used the digitized genetic information from the bacterium Mycoplasma mycoides as a template to build a unique, synthetic genome. To “brand” their work and distinguish it from its genomic template, the team inserted watermarks, retained biologically non-essential polymorphisms, and deleted non-coding regions. This novel genome, named M. mycoides JCVI-syn1.0, was then transplanted into the bacterium Mycoplasma capricolum, where it eventually replaced the cell’s protein infrastructure and replicated itself, becoming the new, synthetic M. mycoides species.

This is synthetic genomics: the idea that the genetic code is the software of an organism, and the proteins that compose the cell its hardware. However, in this system, new software that codes for the manufacture and delivery of its own custom hardware can be “uploaded”.

Methods

Synthetic Genome Assembly Strategy

How did they do this? A hierarchical strategy was employed to assemble the complete synthetic genome in three stages, in vivo, using the yeast, Saccharomyces cerevisiae, as the staging-host and recombination workhorse. The genome was assembled from 1078 overlapping cassettes – each approximately 1080 base pairs (bp) in length – in three steps:

  1. 1080-bp cassettes with 80-bp overlaps were individually assembled from chemically synthesized oligonucleotides and then sequence-verified by Blue Heron [Figure 1, orange arrows];
  2. These cassettes were assembled in sets of 10 to produce 109 10-kbp assemblies ;Figure 1, blue arrows];
  3. The intermediate assemblies were further combined in sets of 10 to produce 11 100-kbp macro-assemblies; to proceed beyond this point, it was necessary to generate microgram quantities of each of the 11 “green arrow” assemblies. Once isolated as circular plasmids, they were treated with exonuclease and passed through an anion-exchange column; to further screen out linear yeast chromosomal DNA, agarose gels were used to “trap” the circular plasmids while allowing the linear yeast DNA to be pulled  through using electrophoresis [Figure 1, green arrows];
  4. The 11 “green arrow” assemblies were finally recombined as the complete genome [Figure 1, red circle];
  5. At each assembly stage, Multiplex polymerase chain reaction (mPCR) was used to confirm the presence and completeness of all constitutive parts; because each intermediate component had its own primer pair, the presence, at each stage, of all appropriate amplicons suggested that that stage of assembly had been successful;
  6. All constructs were assembled by in vivo homologous recombination in yeast, with two exceptions, which were enzymatically joined in vitro [Figure 1, white arrows];
  7. Major variations from template genome: watermarks (Figure 1, WM1 – 4); 94-D (4-kbp region intentionally deleted); and yeast growth and transplantation elements [Figure 1, yellow circles];
  8. There are also 20 nucleotide polymorphisms [Figure 1, asterisks]

    Visualization of Methods

    Figure 1: Visualization of Genome Assembly Procedure

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Results

Finally, transplantation of the synthetic genome from the intact yeast cells into the Mycoplasma capricolum bacterium host was accomplished by a process described by Dr. Carole Latrigue in 2009 and was not elucidated in this report. The team also inserted a lacZ gene into JCVI-syn1.0 that, in response to tetracycline and X-gal, turns colonies blue.

After transplantation of the synthetic genome, the team used multiplex PCR to distinguish it from m. mycoides by specifically testing for the encoded watermarks. Finally, the genome was sequenced and found to match the intended design with a few exceptions: 8 new polymorphisms, a transposon insertion, and an 85-bp duplication. No sequenced genes were in common with M. capricolum, indicating that its genome was completely replaced by that of JCVI-syn1.0.

Discussion

What do these results mean? Concisely: success. This is high-caliber, smart, brave science, at once basic and applied, and built upon years of painstakingly small, progressive steps. The synthetic genome contains segments — watermarks, one of which bears the words of the famous physicist Richard Feynman “What I cannot build, I cannot understand”.

What is the next step? The team at JCVI is already working on it; to synthesize a minimal cell that has only the machinery necessary for independent life. This minimal cell will enable a greater understanding of the function of every gene in a cell and a new vision of cells as understandable machines comprised of biological parts of known function.

Where may this technology take us? The ability to write the software of life is certain to bring with it radical implications for the ways we approach civilization’s most pressing problems. Cells could potentially be designed to produce advanced biofuels and sequester carbon, to purify water, to excrete vaccines and medicines, to manufacture nanomaterials, not to mention all the applications that have yet to be imagined. Perhaps, with the sequencing of the neandertal genome, we could resurrect our nearest kin, or make Jurassic Park a reality. But, this is also a potentially dangerous technology with which our ethics will inevitably struggle to keep pace.

Title of Article: Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome

Authors: Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang, Mikkel A. Algire, Gwynedd A. Benders, Michael G. Montague, Li Ma, Monzia M. Moodie, Chuck Merryman, Sanjay Vashee, Radha Krishnakumar, Nacyra Assad-Garcia, Cynthia Andrews-Pfannkoch, Evgeniya A. Denisova, Lei Young, Zhi-Qing Qi, Thomas H. Segall-Shapiro, Christopher H. Calvey, Prashanth P. Parmar, Clyde A. Hutchison III, Hamilton O. Smith and J. Craig Venter

Institutional affiliations: The J. Craig Venter Institute, Rockville MD and San Diego, CA

Point of Publication: Published Online 20 May 2010 / Science, 2 July 2010: Vol. 329 no. 5987 pp. 52-5

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