Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome

@article{Gibson2008CompleteCS,
  title={Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome},
  author={Daniel G. Gibson and Gwynedd A. Benders and Cynthia Andrews-Pfannkoch and Evgeniya A. Denisova and Holly Baden-Tillson and Jayshree Zaveri and Timothy B. Stockwell and Anushka Brownley and David W. Thomas and Mikkel A. Algire and Chuck Merryman and Lei Young and V. N. Noskov and John I. Glass and J. Craig Venter and Clyde A. Hutchison and Hamilton O. Smith},
  journal={Science},
  year={2008},
  volume={319},
  pages={1215 - 1220}
}
We have synthesized a 582,970–base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted “watermarks” at intergenic sites known to tolerate transposon insertions. Overlapping “cassettes” of 5 to 7 kilobases (kb), assembled from chemically synthesized… 
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The first month of 2008 was unusually quiet in terms of microbial genome sequencing, but the announcement by J. Craig Venter and colleagues that they ‘have synthesized a 582 970 bp Mycoplasma genitalium genome’ suggests the era of synthetic biology has officially begun.
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References

SHOWING 1-10 OF 56 REFERENCES
Generating a synthetic genome by whole genome assembly: φX174 bacteriophage from synthetic oligonucleotides
TLDR
Conditions are established for the rapid assembly of the complete infectious genome of bacteriophage φX174 from a single pool of chemically synthesized oligonucleotides and sequence analysis of several infectious isolates verified the accuracy of these synthetic genomes.
Global transposon mutagenesis and a minimal Mycoplasma genome.
TLDR
Global transposon mutagenesis was used to identify nonessential genes in an effort to learn whether the naturally occurring gene complement is a true minimal genome under laboratory growth conditions, and suggests that 265 to 350 of the 480 protein-coding genes of M. genitalium are essential under laboratory growing conditions.
Combining two genomes in one cell: stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome.
TLDR
Given the significant diversity in genome structure observed upon horizontal DNA transfer in nature, this stable laboratory-generated composite genome raised fundamental questions concerning two complete genomes in one cell and may be generally applicable to other genomes or genome loci of free-living organisms.
The Minimal Gene Complement of Mycoplasma genitalium
TLDR
Comparison of the Mycoplasma genitalium genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.
Segments missing from the draft human genome sequence can be isolated by transformation‐associated recombination cloning in yeast
TLDR
Re‐sequencing of two human genomic fragments re‐isolated from human DNA by transformation‐associated recombination (TAR) cloning showed that TAR cloning might be a valuable method that could be widely used during the final stages of the Human Genome Project.
Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59
TLDR
A novel method was developed to assemble a full-length infectious cDNA of the group II coronavirus mouse hepatitis virus strain A59, which has the potential to be used to construct viral, microbial, or eukaryotic genomes approaching several million base pairs in length and used to insert restriction sites at any given nucleotide in a microbial genome.
Essential genes of a minimal bacterium.
TLDR
Using global transposon mutagenesis, this work identifies 382 of the 482 M. genitalium protein-coding genes as essential, plus five sets of disrupted genes that encode proteins with potentially redundant essential functions, such as phosphate transport.
Optimum conditions for selective isolation of genes from complex genomes by transformation-associated recombination cloning.
TLDR
It is demonstrated here that the transformant yield increases approximately 10-fold when the genomic DNA is sheared to 100-200 kb before being presented to the spheroplasts, and that the targeted sequences recombine much more efficiently with the vector's targeting hooks when they are located at the ends of the genomicDNA fragment.
Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster.
TLDR
This work has developed and implemented a strategy for the high-throughput synthesis of long, accurate DNA sequences and validated the approach by building a synthetic 31,656-bp polyketide synthase gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and itspolyketide product in Escherichia coli.
Rebuilding microbial genomes.
TLDR
In conclusion, rebuilding and booting a genome remains a feasible and pragmatic approach to creating a synthetic microbial cell.
...
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