-
Bogumil J Karas,
Jelena Jablanovic,
Lijie Sun,
Li Ma,
Gregory M Goldgof,
Jason Stam,
Adi Ramon,
Micah J Manary,
Elizabeth A Winzeler,
J Craig Venter,
Philip D Weyman,
Daniel G Gibson,
John I Glass,
Clyde A Hutchison, Hamilton O Smith,
Yo Suzuki
[show abstract]
[hide abstract]
ABSTRACT: Transfer of genomes into yeast facilitates genome engineering for genetically intractable organisms, but this process has been hampered by the need for cumbersome isolation of intact genomes before transfer. Here we demonstrate direct cell-to-cell transfer of bacterial genomes as large as 1.8 megabases (Mb) into yeast under conditions that promote cell fusion. Moreover, we discovered that removal of restriction endonucleases from donor bacteria resulted in the enhancement of genome transfer.
Nature Methods 03/2013; · 19.28 Impact Factor
-
Christian Tagwerker,
Christopher L Dupont,
Bogumil J Karas,
Li Ma,
Ray-Yuan Chuang,
Gwynedd A Benders,
Adi Ramon,
Mark Novotny,
Michael G Montague,
Pratap Venepally,
Daniel Brami,
Ariel Schwartz,
Cynthia Andrews-Pfannkoch,
Daniel G Gibson,
John I Glass, Hamilton O Smith,
J Craig Venter,
Clyde A Hutchison
[show abstract]
[hide abstract]
ABSTRACT: 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.
Nucleic Acids Research 08/2012; · 8.03 Impact Factor
-
Vladimir N Noskov,
Bogumil J Karas,
Lei Young,
Ray-Yuan Chuang,
Daniel G Gibson,
Ying-Chi Lin,
Jason Stam,
Isaac T Yonemoto,
Yo Suzuki,
Cynthia Andrews-Pfannkoch,
John I Glass, Hamilton O Smith,
Clyde A Hutchison,
J Craig Venter,
Philip D Weyman
[show abstract]
[hide abstract]
ABSTRACT: The ability to assemble large pieces of prokaryotic DNA by yeast recombination has great application in synthetic biology, but cloning large pieces of high G+C prokaryotic DNA in yeast can be challenging. Additional considerations in cloning large pieces of high G+C DNA in yeast may be related to toxic genes, to the size of the DNA, or to the absence of yeast origins of replication within the sequence. As an example of our ability to clone high G+C DNA in yeast, we chose to work with Synechococcus elongatus PCC 7942, which has an average G+C content of 55%. We determined that no regions of the chromosome are toxic to yeast and that S. elongatus DNA fragments over ∼200 kb are not stably maintained. DNA constructs with a total size under 200 kb could be readily assembled, even with 62 kb of overlapping sequence between pieces. Addition of yeast origins of replication throughout allowed us to increase the total size of DNA that could be assembled to at least 454 kb. Thus, cloning strategies utilizing yeast recombination with large, high G+C prokaryotic sequences should include yeast origins of replication as a part of the design process.
ACS synthetic biology. 07/2012; 1(7):267-73.
-
[show abstract]
[hide abstract]
ABSTRACT: Cloning of whole genomes of the genus Mycoplasma in yeast has been an essential step for the creation of the first synthetic cell. The genome of the synthetic cell is based on Mycoplasma mycoides, which deviates from the universal genetic code by encoding tryptophan rather than the UGA stop codon. The feature was thought to be important because bacterial genes might be toxic to the host yeast cell if driven by a cryptic promoter active in yeast. As we move to expand the range of bacterial genomes cloned in yeast, we extended this technology to bacteria that use the universal genetic code. Here we report cloning of the Acholeplasma laidlawii PG-8A genome, which uses the universal genetic code. We discovered that only one A. laidlawii gene, a surface anchored extracellular endonuclease, was toxic when cloned in yeast. This gene was inactivated in order to clone and stably maintain the A. laidlawii genome as a centromeric plasmid in the yeast cell.
ACS synthetic biology. 01/2012; 1(1):22-8.
-
[show abstract]
[hide abstract]
ABSTRACT: Alteromonas macleodii Deep ecotype is a marine, heterotrophic, gammaproteobacterium isolated in the Mediterranean Sea between depths of 1000 and 3500 m. The sequenced strain was previously reported to contain a [NiFe] hydrogenase. We verified the presence of this hydrogenase in other strains of A. macleodii Deep ecotype that were previously isolated from several bathypelagic microenvironments. We developed a system for the genetic manipulation of A. macleodii Deep ecotype using conjugation and used this system to create mutant strains that lack the [NiFe] hydrogenase structural genes (hynSL). The mutants did not possess hydrogenase activity, and complementation of the mutant strain with the hynSL genes successfully restored hydrogenase activity. Both the mutant and the wild-type strains grew at the same rate in a variety of media and under different environmental conditions, indicating little effect of the hydrogenase mutation under the conditions tested.
FEMS Microbiology Letters 06/2011; 322(2):180-7. · 2.04 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: HynSL from Alteromonas macleodii 'deep ecotype' (AltDE) is an oxygen-tolerant and thermostable [NiFe] hydrogenase. Its two structural genes (hynSL), encoding small and large hydrogenase subunits, are surrounded by eight genes (hynD, hupH and hypCABDFE) predicted to encode accessory proteins involved in maturation of the hydrogenase. A 13 kb fragment containing the ten structural and accessory genes along with three additional adjacent genes (orf2, cyt and orf1) was cloned into an IPTG-inducible expression vector and transferred into an Escherichia coli mutant strain lacking its native hydrogenases. Upon induction, HynSL from AltDE was expressed in E. coli and was active, as determined by an in vitro hydrogen evolution assay. Subsequent genetic analysis revealed that orf2, cyt, orf1 and hupH are not essential for assembling an active hydrogenase. However, hupH and orf2 can enhance the activity of the heterologously expressed hydrogenase. We used this genetic system to compare maturation mechanisms between AltDE HynSL and its Thiocapsa roseopersicina homologue. When the structural genes for the T. roseopersicina hydrogenase, hynSL, were expressed along with known T. roseopersicina accessory genes (hynD, hupK, hypC1C2 and hypDEF), no active hydrogenase was produced. Further, co-expression of AltDE accessory genes hypA and hypB with the entire set of the T. roseopersicina genes did not produce an active hydrogenase. However, co-expression of all AltDE accessory genes with the T. roseopersicina structural genes generated an active T. roseopersicina hydrogenase. This result demonstrates that the accessory genes from AltDE can complement their counterparts from T. roseopersicina and that the two hydrogenases share similar maturation mechanisms.
Microbiology 02/2011; 157(Pt 5):1363-74. · 3.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hydrogenases are enzymes involved in the bioproduction of hydrogen, a clean alternative energy source whose combustion generates water as the only end product. In this article we identified and characterized a [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii "deep ecotype" with unusual stability toward oxygen and high temperature. The A. macleodii hydrogenase (HynSL) can catalyze both H(2) evolution and H(2) uptake reactions. HynSL was expressed in A. macleodii under aerobic conditions and reached the maximum activity when the cells entered the late exponential phase. The higher level of hydrogenase activity was accompanied by a greater abundance of the HynSL protein in the late-log or stationary phase. The addition of nickel to the growth medium significantly enhanced the hydrogenase activity. Ni treatment affected the level of the protein, but not the mRNA, indicating that the effect of Ni was exerted at the posttranscriptional level. Hydrogenase activity was distributed ∼30% in the membrane fraction and ∼70% in the cytoplasmic fraction. Thus, HynSL appears to be loosely membrane-bound. Partially purified A. macleodii hydrogenase demonstrated extraordinary stability. It retained 84% of its activity after exposure to 80°C for 2 h. After exposure to air for 45 days at 4°C, it retained nearly 100% of its activity when assayed under anaerobic conditions. Its catalytic activity in the presence of O(2) was evaluated by the hydrogen-deuterium (H-D) exchange assay. In 1% O(2), 20.4% of its H-D exchange activity was retained. The great stability of HynSL makes it a potential candidate for biotechnological applications.
Applied and environmental microbiology 01/2011; 77(6):1990-8. · 3.69 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H(2) evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria.
PLoS ONE 01/2011; 6(5):e20126. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We describe a one-step, isothermal assembly method for synthesizing DNA molecules from overlapping oligonucleotides. The method cycles between in vitro recombination and amplification until the desired length is reached. As a demonstration of its simplicity and robustness, we synthesized the entire 16.3-kilobase mouse mitochondrial genome from 600 overlapping 60-mers.
Nature Methods 10/2010; 7(11):901-3. · 19.28 Impact Factor
-
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, [......],
Cynthia Andrews-Pfannkoch,
Evgeniya A Denisova,
Lei Young,
Zhi-Qing Qi,
Thomas H Segall-Shapiro,
Christopher H Calvey,
Prashanth P Parmar,
Clyde A Hutchison, Hamilton O Smith,
J Craig Venter
[show abstract]
[hide abstract]
ABSTRACT: 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.
Science 07/2010; 329(5987):52-6. · 31.20 Impact Factor
-
Gwynedd A Benders,
Vladimir N Noskov,
Evgeniya A Denisova,
Carole Lartigue,
Daniel G Gibson,
Nacyra Assad-Garcia,
Ray-Yuan Chuang,
William Carrera,
Monzia Moodie,
Mikkel A Algire,
Quang Phan,
Nina Alperovich,
Sanjay Vashee,
Chuck Merryman,
J Craig Venter, Hamilton O Smith,
John I Glass,
Clyde A Hutchison
[show abstract]
[hide abstract]
ABSTRACT: 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.
Nucleic Acids Research 03/2010; 38(8):2558-69. · 8.03 Impact Factor
-
Carole Lartigue,
Sanjay Vashee,
Mikkel A Algire,
Ray-Yuan Chuang,
Gwynedd A Benders,
Li Ma,
Vladimir N Noskov,
Evgeniya A Denisova,
Daniel G Gibson,
Nacyra Assad-Garcia,
Nina Alperovich,
David W Thomas,
Chuck Merryman,
Clyde A Hutchison, Hamilton O Smith,
J Craig Venter,
John I Glass
[show abstract]
[hide abstract]
ABSTRACT: 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.
Science 09/2009; 325(5948):1693-6. · 31.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a Thiocapsa roseopersicina O2-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to the hyaA and hyaB genes of an Alteromonas macleodii hydrogenase, indicating that it belongs to the Alteromonas clade. We were able to express our new Alteromonas hydrogenase in T. roseopersicina. Expression was accomplished by coexpressing only two accessory genes, hyaD and hupH, without the need to express any of the hyp accessory genes (hypABCDEF). These results suggest that the native accessory proteins in T. roseopersicina could substitute for the Alteromonas counterparts that are absent in the host to facilitate the assembly of a functional Alteromonas hydrogenase. To further compare the complex assembly machineries of these two [NiFe] hydrogenases, we performed complementation experiments by introducing the new Alteromonas hyaD gene into the T. roseopersicina hynD mutant. Interestingly, Alteromonas endopeptidase HyaD could complement T. roseopersicina HynD to cleave endoproteolytically the C-terminal end of the T. roseopersicina HynL hydrogenase large subunit and activate the enzyme. This study refines our knowledge on the selectivity and pleiotropy of the elements of the [NiFe] hydrogenase assembly machineries. It also provides a model for functionally analyzing novel enzymes from environmental microbes in a culture-independent manner.
Applied and environmental microbiology 08/2009; 75(18):5821-30. · 3.69 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
Nature Methods 05/2009; 6(5):343-5. · 19.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Proceedings of the National Academy of Sciences 01/2009; 105(51):20404-9. · 9.68 Impact Factor
-
Molecular Systems Biology 01/2009; 5:330. · 8.63 Impact Factor
-
Daniel G Gibson,
Gwynedd A Benders,
Cynthia Andrews-Pfannkoch,
Evgeniya A Denisova,
Holly Baden-Tillson,
Jayshree Zaveri,
Timothy B Stockwell,
Anushka Brownley,
David W Thomas,
Mikkel A Algire,
Chuck Merryman,
Lei Young,
Vladimir N Noskov,
John I Glass,
J Craig Venter,
Clyde A Hutchison, Hamilton O Smith
[show abstract]
[hide abstract]
ABSTRACT: 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.
Science 03/2008; 319(5867):1215-20. · 31.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: As a step toward propagation of synthetic genomes, we completely replaced the genome of a bacterial cell with one from another species by transplanting a whole genome as naked DNA. Intact genomic DNA from Mycoplasma mycoides large colony (LC), virtually free of protein, was transplanted into Mycoplasma capricolum cells by polyethylene glycol-mediated transformation. Cells selected for tetracycline resistance, carried by the M. mycoides LC chromosome, contain the complete donor genome and are free of detectable recipient genomic sequences. These cells that result from genome transplantation are phenotypically identical to the M. mycoides LC donor strain as judged by several criteria.
Science 09/2007; 317(5838):632-8. · 31.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mycoplasma genitalium has the smallest genome of any organism that can be grown in pure culture. It has a minimal metabolism and little genomic redundancy. Consequently, its genome is expected to be a close approximation to the minimal set of genes needed to sustain bacterial life. Using global transposon mutagenesis, we isolated and characterized gene disruption mutants for 100 different nonessential protein-coding genes. None of the 43 RNA-coding genes were disrupted. Herein, we identify 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. Genes encoding proteins of unknown function constitute 28% of the essential protein-coding genes set. Disruption of some genes accelerated M. genitalium growth.
Proceedings of the National Academy of Sciences 02/2006; 103(2):425-30. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We describe conditions for rolling-circle amplification (RCA) of individual DNA molecules 5–7 kb in size by >109-fold, using φ29 DNA polymerase. The principal difficulty with amplification of small amounts of template by RCA using φ29
DNA polymerase is “background” DNA synthesis that usually occurs when template is omitted, or at low template concentrations.
Reducing the reaction volume while keeping the amount of template fixed increases the template concentration, resulting in
a suppression of background synthesis. Cell-free cloning of single circular molecules by using φ29 DNA polymerase was achieved
by carrying out the amplification reactions in very small volumes, typically 600 nl. This procedure allows cell-free cloning
of individual synthetic DNA molecules that cannot be cloned in Escherichia coli, for example synthetic phage genomes carrying lethal mutations. It also allows cell-free cloning of genomic DNA isolated
from bacteria. This DNA can be sequenced directly from the φ29 DNA polymerase reaction without further amplification. In contrast
to PCR amplification, RCA using φ29 DNA polymerase does not produce mutant jackpots, and the high processivity of the enzyme
eliminates stuttering at homopolymer tracts. Cell-free cloning has many potential applications to both natural and synthetic
DNA. These include environmental DNA samples that have proven difficult to clone and synthetic genes encoding toxic products.
The method may also speed genome sequencing by eliminating the need for biological cloning.
Proceedings of the National Academy of Sciences 11/2005; 102(48):17332-17336. · 9.68 Impact Factor
