Gwynedd A. Benders

CREATION OF A BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME

Let There Be Life The DNA sequence information from thousands of genomes is stored digitally as ones and zeros in computer memory. Now, Gibson et al. (p. 52, published online 20 May; see the cover; see the Policy Forum by Cho and Relman) have brought together technologies from the past 15 years to start from digital information on the genome of Mycoplasma mycoides to chemically synthesize the genomic DNA as segments that could then be assembled in yeast and transplanted into the cytoplasm of another organism. A number of methods were also incorporated to facilitate testing and error correction of the synthetic genome segments. The transplanted genome became established in the recipient cell, replacing the recipient genome, which was lost from the cell. The reconstituted cells were able to replicate and form colonies, providing a proof-of-principle for future developments in synthetic biology. A synthetic Mycoplasma mycoides genome transplanted into M. capricolum was able to control the host cell. We report 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 M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including “watermark” sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.

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

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 oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb (“1/8 genome”), and 144 kb (“1/4 genome”), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.

One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome.

We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments.

Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast

Character Transplant When engineering bacteria, it can be advantageous to propagate the genomes in yeast. However, to be truly useful, one must be able to transplant the bacterial chromosome from yeast back into a recipient bacterial cell. But because yeast does not contain restriction-modification systems, such transplantation poses problems not encountered in transplantation from one bacterial cell to another. Bacterial genomes isolated after growth in yeast are likely to be susceptible to the restriction-modification system(s) of the recipient cell, as well as their own. Lartigue et al. (p. 1693, published online 20 August) describe multiple steps, including in vitro DNA methylation, developed to overcome such barriers. A Mycoplasma mycoides large-colony genome was propagated in yeast as a centromeric plasmid, engineered via yeast genetic systems, and, after specific methylation, transplanted into M. capricolum to produce a bacterial cell with the genotype and phenotype of the altered M. mycoides large-colony genome. A Mycoplasma mycoides genome was engineered in yeast and then transplanted into M. capricolum cells to produce a new strain. We recently reported the chemical synthesis, assembly, and cloning of a bacterial genome in yeast. To produce a synthetic cell, the genome must be transferred from yeast to a receptive cytoplasm. Here we describe methods to accomplish this. We cloned a Mycoplasma mycoides genome as a yeast centromeric plasmid and then transplanted it into Mycoplasma capricolum to produce a viable M. mycoides cell. While in yeast, the genome was altered by using yeast genetic systems and then transplanted to produce a new strain of M. mycoides. These methods allow the construction of strains that could not be produced with genetic tools available for this bacterium.

Cloning whole bacterial genomes in yeast

Most microbes have not been cultured, and many of those that are cultivatable are difficult, dangerous or expensive to propagate or are genetically intractable. Routine cloning of large genome fractions or whole genomes from these organisms would significantly enhance their discovery and genetic and functional characterization. Here we report the cloning of whole bacterial genomes in the yeast Saccharomyces cerevisiae as single-DNA molecules. We cloned the genomes of Mycoplasma genitalium (0.6 Mb), M. pneumoniae (0.8 Mb) and M. mycoides subspecies capri (1.1 Mb) as yeast circular centromeric plasmids. These genomes appear to be stably maintained in a host that has efficient, well-established methods for DNA manipulation.

Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast

Marine cyanobacteria of the genus Prochlorococcus represent numerically dominant photoautotrophs residing throughout the euphotic zones in the open oceans and are major contributors to the global carbon cycle. Prochlorococcus has remained a genetically intractable bacterium due to slow growth rates and low transformation efficiencies using standard techniques. Our recent successes in cloning and genetically engineering the AT-rich, 1.1 Mb Mycoplasma mycoides genome in yeast encouraged us to explore similar methods with Prochlorococcus. Prochlorococcus MED4 has an AT-rich genome, with a GC content of 30.8%, similar to that of Saccharomyces cerevisiae (38%), and contains abundant yeast replication origin consensus sites (ACS) evenly distributed around its 1.66 Mb genome. Unlike Mycoplasma cells, which use the UGA codon for tryptophane, Prochlorococcus uses the standard genetic code. Despite this, we observed no toxic effects of several partial and 15 whole Prochlorococcus MED4 genome clones in S. cerevisiae. Sequencing of a Prochlorococcus genome purified from yeast identified 14 single base pair missense mutations, one frameshift, one single base substitution to a stop codon and one dinucleotide transversion compared to the donor genomic DNA. We thus provide evidence of transformation, replication and maintenance of this 1.66 Mb intact bacterial genome in S. cerevisiae.

Targeted Chromosomal Knockouts in Mycoplasma pneumoniae

ABSTRACT Most gene knockouts in mycoplasmas are achieved through labor-intensive transposon mutagenesis. Here, we describe a method for making targeted deletions in Mycoplasma pneumoniae by use of homologous recombination. In this method, M. pneumoniae is transformed with a plasmid carrying an antibiotic resistance marker flanked by 1-kb regions surrounding the target gene. Following selection for the antibiotic resistance, colonies are screened for double crossovers which indicate complete deletion of the target open reading frame.