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

  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},
  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… 

The dawn of synthetic genomics

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.

Genomic Iterative Replacements of Large Synthetic DNA Fragments in Corynebacterium glutamicum.

A genomic iterative replacement system based on RecET recombination for C. glutamicum, involving the successive integration of up to 10 kb DNA fragments obtained in vitro, and the transformants are selected by the alternative use of kanR and speR selectable markers, which can be used for the synthesis of a larger region of the genome.

Cloning, Stability, and Modification of Mycoplasma hominis Genome in Yeast.

The successful one-step cloning of the M. hominis PG21 genome in yeast using the transformation-associated recombination (TAR) cloning method and the CRISPR/Cas9 editing tool, available in Saccharomyces cerevisiae are reported.

Mycoplasma pneumoniae Genome Editing Based on Oligo Recombineering and Cas9-Mediated Counterselection

An oligonucleotide recombineering method based on GP35, a ssDNA recombinase originally encoded by a Bacillus subtilis-associated phage, is developed, envisioning this technology as a major step toward the use of M. pneumoniae, and possibly other Mycoplasmas, as synthetic biology chassis strains.

Chemical Synthesis of Bacteriophage G4

This study demonstrated that a phage can be synthesized and manipulated genetically according to the sequences, and can be efficient enough to infect the Escherichia coli, showing the potential use of synthetic biology in medical application.

Mega-cloning and the advent of synthetic genomes

One novel branch of recombinant DNA, referred to as synthetic genomics, is occupied with (re)-construction of entire cellular genomes from virtual sequence information and using chemical components.

Synthetic Genomics: From DNA Synthesis to Genome Design.

Using bottom-up assembly and applying genome-wide alterations will improve the understanding of genome structure and function and provide a platform for systematic studies of eukaryotic chromosomes.

Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome

The design, synthesis, and assembly of the 1.08–mega–base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new cells that are controlled only by the synthetic chromosome are reported.

Tandem repeat coupled with endonuclease cleavage (TREC): a seamless modification tool for genome engineering in yeast

A method that can seamlessly modify the bacterial genome in yeast with high efficiency is reported, which can be adapted for a variety of genomic modifications and may provide an important tool to modify and design natural or synthetic genomes propagated in yeast.



Generating a synthetic genome by whole genome assembly: φX174 bacteriophage from synthetic oligonucleotides

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.

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.

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

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

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

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.

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.

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.

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.

In conclusion, rebuilding and booting a genome remains a feasible and pragmatic approach to creating a synthetic microbial cell.