Nacyra Assad-Garcia

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.

A Whole-Cell Computational Model Predicts Phenotype from Genotype

Understanding how complex phenotypes arise from individual molecules and their interactions is a primary challenge in biology that computational approaches are poised to tackle. We report a whole-cell computational model of the life cycle of the human pathogen Mycoplasma genitalium that includes all of its molecular components and their interactions. An integrative approach to modeling that combines diverse mathematics enabled the simultaneous inclusion of fundamentally different cellular processes and experimental measurements. Our whole-cell model accounts for all annotated gene functions and was validated against a broad range of data. The model provides insights into many previously unobserved cellular behaviors, including in vivo rates of protein-DNA association and an inverse relationship between the durations of DNA replication initiation and replication. In addition, experimental analysis directed by model predictions identified previously undetected kinetic parameters and biological functions. We conclude that comprehensive whole-cell models can be used to facilitate biological discovery.

Design and synthesis of a minimal bacterial genome

Designing and building a minimal genome A goal in biology is to understand the molecular and biological function of every gene in a cell. One way to approach this is to build a minimal genome that includes only the genes essential for life. In 2010, a 1079-kb genome based on the genome of Mycoplasma mycoides (JCV-syn1.0) was chemically synthesized and supported cell growth when transplanted into cytoplasm. Hutchison III et al. used a design, build, and test cycle to reduce this genome to 531 kb (473 genes). The resulting JCV-syn3.0 retains genes involved in key processes such as transcription and translation, but also contains 149 genes of unknown function. Science, this issue p. 10.1126/science.aad6253 Cycles of design, building, and testing produced a 531-kilobase genome comprising 473 genes. INTRODUCTION In 1984, the simplest cells capable of autonomous growth, the mycoplasmas, were proposed as models for understanding the basic principles of life. In 1995, we reported the first complete cellular genome sequences (Haemophilus influenza, 1815 genes, and Mycoplasma genitalium, 525 genes). Comparison of these sequences revealed a conserved core of about 250 essential genes, much smaller than either genome. In 1999, we introduced the method of global transposon mutagenesis and experimentally demonstrated that M. genitalium contains many genes that are nonessential for growth in the laboratory, even though it has the smallest genome known for an autonomously replicating cell found in nature. This implied that it should be possible to produce a minimal cell that is simpler than any natural one. Whole genomes can now be built from chemically synthesized oligonucleotides and brought to life by installation into a receptive cellular environment. We have applied whole-genome design and synthesis to the problem of minimizing a cellular genome. RATIONALE Since the first genome sequences, there has been much work in many bacterial models to identify nonessential genes and define core sets of conserved genetic functions, using the methods of comparative genomics. Often, more than one gene product can perform a particular essential function. In such cases, neither gene will be essential, and neither will necessarily be conserved. Consequently, these approaches cannot, by themselves, identify a set of genes that is sufficient to constitute a viable genome. We set out to define a minimal cellular genome experimentally by designing and building one, then testing it for viability. Our goal is a cell so simple that we can determine the molecular and biological function of every gene. RESULTS Whole-genome design and synthesis were used to minimize the 1079–kilobase pair (kbp) synthetic genome of M. mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology in combination with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three more cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kbp, 473 genes). Its genome is smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 has a doubling time of ~180 min, produces colonies that are morphologically similar to those of JCVI-syn1.0, and appears to be polymorphic when examined microscopically. CONCLUSION The minimal cell concept appears simple at first glance but becomes more complex upon close inspection. In addition to essential and nonessential genes, there are many quasi-essential genes, which are not absolutely critical for viability but are nevertheless required for robust growth. Consequently, during the process of genome minimization, there is a trade-off between genome size and growth rate. JCVI-syn3.0 is a working approximation of a minimal cellular genome, a compromise between small genome size and a workable growth rate for an experimental organism. It retains almost all the genes that are involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions, suggesting the presence of undiscovered functions that are essential for life. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design. Four design-build-test cycles produced JCVI-syn3.0. (A) The cycle for genome design, building by means of synthesis and cloning in yeast, and testing for viability by means of genome transplantation. After each cycle, gene essentiality is reevaluated by global transposon mutagenesis. (B) Comparison of JCVI-syn1.0 (outer blue circle) with JCVI-syn3.0 (inner red circle), showing the division of each into eight segments. The red bars inside the outer circle indicate regions that are retained in JCVI-syn3.0

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.

A Structurally Distinct Human Mycoplasma Protein that Generically Blocks Antigen-Antibody Union

Easy M Our immune systems can produce a vastly diverse repertoire of antibody molecules that each recognize and bind to a specific foreign antigen via a hypervariable region. However, there are a few bacterial antigens—such as Protein A, Protein G, and Protein L—that instead bind to the antibodys conserved regions and can bind to a large number of different antibodies. These high-affinity broad-spectrum antibody-binding properties have been widely exploited both in the laboratory and in industry for purifying, immobilizing, and detecting antibodies. Grover et al. (p. 656) have now identified Protein M found on the surface of human mycoplasma, which displays even broader antibody-binding specificity. The crystal structure of Protein M revealed how Protein-M binding blocks the antibodys antigen binding site. This mechanism may be exploited by mycoplasma to escape the humoral immune response. High-affinity binding of Protein M to a very broad range of human antibodies may find widespread immunochemical applications. We report the discovery of a broadly reactive antibody-binding protein (Protein M) from human mycoplasma. The crystal structure of the ectodomain of transmembrane Protein M differs from other known protein structures, as does its mechanism of antibody binding. Protein M binds with high affinity to all types of human and nonhuman immunoglobulin G, predominantly through attachment to the conserved portions of the variable region of the κ and λ light chains. Protein M blocks antibody-antigen union, likely because of its large C-terminal domain extending over the antibody-combining site, blocking entry to large antigens. Similar to the other immunoglobulin-binding proteins such as Protein A, Protein M as well as its orthologs in other Mycoplasma species could become invaluable reagents in the antibody field.

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.

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼ 10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.

New Selectable Marker for Manipulating the Simple Genomes of Mycoplasma Species

ABSTRACT Over the past several years, significant advances have been made in the molecular genetics of the Mollicutes (the simplest cells that can be grown in axenic culture). Nevertheless, a number of basic molecular tools are still required before genetic manipulations become routine. Here we describe the development of a new dominant selectable marker based on the enzyme puromycin-N-acetyltransferase from Streptomyces alboniger. Puromycin is an antibiotic that mimics the 3′-terminal end of aminoacylated tRNAs and attaches to the carboxyl terminus of growing protein chains. This stops protein synthesis. Because puromycin conscripts rRNA recognition elements that are used by all of the various tRNAs in a cell, it is unlikely that spontaneous antibiotic resistance can be acquired via a simple point mutation—an annoying issue with existing mycoplasma markers. Our codon-optimized cassette confers pronounced puromycin resistance on all five of the mycoplasma species we have tested so far. The resistance cassette was also designed to function in Escherichia coli, which simplifies the construction of shuttle vectors and makes it trivial to produce the large quantities of DNA generally necessary for mycoplasma transformation. Due to these and other features, we expect the puromycin marker to be a widely applicable tool for studying these simple cells and pathogens.

Enhancement of targeted homologous recombination in Mycoplasma mycoides subsp. capri by inclusion of heterologous recA.

ABSTRACT A suicide plasmid, pExp1-ctpA::tetM-recAec, employing recA from Escherichia coli and tetM as a selection marker, was used to generate ctpA knockout mutants in Mycoplasma mycoides subsp. capri through targeted gene disruption. Inclusion of E. coli recA greatly enhanced both the consistency and the recovery of mutants generated by homologous recombination.