(Post no Facebook em 10 (out)/31/2016)
(Post no Facebook em 10 (out)/31/2016)
Interessante artigo informando varias opinioes inteligentes sobre esse grande misterio e a seguir, minha opiniao registrada no comentario abaixo:
Meu comentario postado no artigo:
Pois a minha investigação pessoal com varias experiencias incomuns me conduziram a uma diferente ideia sobre o que se postula como “alma”. As ondas de luz naturais imitam o ciclo vital do nosso corpo. Portanto, uma onda de luz natural foi a primeira forma “vital”, a primeira forma de sistema natural funcional neste mundo material. A luz e’ composta de partículas, fótons, os quais, trouxeram as informações deste sistema astronomico que produziu as formas biológicas ( vivos) neste planeta. Ao se encontrarem aqui, estes fótons remontam o sistema de onde vieram, numa rede de conexões, dirigindo os átomos a formarem os corpos vivos físicos. Acontece que esta rede e’ igual ao que os orientais antigos desenharam e chamaram de aura. Assim, a substancia da aura ‘e a luz a qual contem o código da vida. Em outras palavras, neste Universo esta ocorrendo um processo de reprodução da “coisa” que gerou o universo, e o DNA daquela “coisa” e’ uma Matrix/DNA universal, cuja forma atual, na especie humana, e’ a forma da aura. As sinapses dos neurônios se refletem numa nuvem luminosa a qual ainda não podemos perceber, porem esta luz e’ o feto ou embrião de consciência da luz que veio do alem. Claro, isto ainda ‘e uma teoria, estou trabalhando na tentativa de captar esta rede de fótons, provar que átomos e galaxias tiveram suas formas de DNA-aura, etc. No meu website tem as figuras da onda de luz como código da vida, do DNA dos sistemas não-vivos ancestrais, etc. Mas para mim isto faz mais sentido racional que todas explicações acima. Ao menos, enquanto as opiniões no artigo ainda são jogos de palavras, na Toeira da Matrix/DNA apresentamos as imagens, as figuras e coisas palpáveis que podem serem testadas cientificamente. Nos somos imortais, porem numa dimensão em que ainda somos como embriões, nem abrimos os olhos para vê-la, e’ o que sugere esta visão do mundo. Ela sugere que neste planeta cada ser humano tem uma especie de bolha luminosa contendo um gene de uma super-consciência cósmica, mas por enquanto estas “bolhas” estão separadas formando uma camada aureolar superficial de inconsciente coletivo ao redor da Terra, e quando estas bolhas se fundirem numa so, sera’ a consciência do super-organismo de Gaia, a ser fundida com as demais consciências de outros planetas deste Universo. No fim seremos um como o Um de onde viemos, assim como o baby e’ um vindo do humano que o gerou.
Site Title: The Universal Matrix
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Jack Szostak: Reconstructing the First Cells
Daniel G. Gibson, et al. 2010. Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. Science 329:52–56
CREATION OF A BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME
O link para o pdf abaixo veio de:
Life after the synthetic cell (pdf)
Published online 2011 May 25. doi: 10.1186/1471-2148-11-140
( Ler o artigo, depois traduzi-lo)
The last universal common ancestor represents the primordial cellular organism from which diversified life was derived.
O ultimo universal comum ancestral representa o organismo celular primordial a partir do qual a diversificação da vida foi derivada.
(Este artigo não tenho acesso. Copiado aqui o endereço e abstrato para traduzir)
The origin of life has puzzled molecular scientists for over half a century. Yet fundamental questions remain unanswered, including which came first, the metabolic machinery or the encoding nucleic acids. In this study we take a protein-centric view and explore the ancestral origins of proteins. Protein domain structures in proteomes are highly conserved and embody molecular functions and interactions that are needed for cellular and organismal processes. Here we use domain structure to study the evolution of molecular function in the protein world. Timelines describing the age and function of protein domains at fold, fold superfamily, and fold family levels of structural complexity were derived from a structural phylogenomic census in hundreds of fully sequenced genomes. These timelines unfold congruent hourglass patterns in rates of appearance of domain structures and functions, functional diversity, and hierarchical complexity, and revealed a gradual build up of protein repertoires associated with metabolism, translation and DNA, in that order. The most ancient domain architectures were hydrolase enzymes and the first translation domains had catalytic functions for the aminoacylation and the molecular switch-driven transport of RNA. Remarkably, the most ancient domains had metabolic roles, did not interact with RNA, and preceded the gradual build-up of translation. In fact, the first translation domains had also a metabolic origin and were only later followed by specialized translation machinery. Our results explain how the generation of structure in the protein world and the concurrent crystallization of translation and diversified cellular life created further opportunities for proteomic diversification.
Esta experiencia cientifica esta sendo amplamente alardeada e interpretada como a primeira criacao real da vida em laboratorio. Os ateus comemoram afirmando que isto prova que a vida veio da matéria sem vida. Realmente foi um extraordinário e louvável trabalho, e dirigido pela visão de mundo acadêmica atual. Se ela estiver correta, não destruirá a minha teoria da Matrix/DNA, pois esta tambem prevê isso. Porem sera’ um baque `a minha teoria porque eu teria maior dificuldade em provar que a Matrix esta’ inserida de alguma maneira no organismo fabricado.
Eu não entendo todo o escopo contido na célula viva que eles sintetizaram, nem assisti os métodos e processos para ter certeza que foi tudo natural, ou seja, sem usar nada de células vivas para montar esta artificialmente sintetizada. Mas, mesmo sendo leigo, suspeito que um detalhe do método teria sido um erro crasso inutilizando a proposta de criar a vida artificialmente, e o detalhe esta’ no paragrafo do “paper”:
In the first stage, cassettes and a vector were recombined in yeast and transferred to Escherichia coli.50 Plasmid DNAwas then isolated from individual E. coli clones and digested to screen for cells containing a vector with an assembled 10-kb insert.
Primeiro estagio entendo que foi a fase de montagem do genoma sintético, sua inserção num micro-organismo e sua transferência para outro.
Recombinado em yeast (bacteria do fermento) e transferido para E. Coli…?! Ora isto não é produção artificial de vida, de forma alguma. Aqui estão produzindo sim, uma forma de vida, porem, dentro de um ser vivo! Qualquer ser vivo faz isto sozinho e chama-se reprodução. Para se criar vida totalmente artificial, que imite e prove como a vida foi criada na Terra primitiva, tem-se que antes de tudo, reproduzir as condições ambientais da Terra primitiva e somente neste ambiente, copiar, digitalizar e sintetizar o genoma, e outros aparatos iniciais do microorganismo, ate finalmente completa-lo e acompanhar se apos isto a nova montagem vive, se reproduz, etc., por si so.
A Teoria da Matrix/DNA sugere que bits-informação na forma de fótons vindos do Sol e do nucleo terrestre trabalham como genes do sistema astronomico, infiltrando-se nos elétrons dos atomos e dirigindo estes as combinações orgânicas ate a formação de uma célula viva completa. Talvez os cientistas tenham usados átomos contaminados por estes fotons, e com isso, a direcao que deram aos processos funcionou em paralelo as instruções dos fótons nestes átomos. Mas de uma coisa a teoria sugere com mais certeza: todo material de qualquer ser vivo contem os fótons. Portanto, entrar dentro de um corpo vivo e se acercar destes fótons por todos os lados e seras penetrados por eles. E o que fizeram? Inseriram um genoma artificial feitos com átomos, moléculas, iguaizinhos ao genoma natural dentro de um organismo vivo. Se eu entendi direito o paper, esta vida não foi criada em laboratorio mas sim foi reproduzida dentro de um ser vivo.
Hummm… mais abaixo ( na Discussao) o paper diz:
We refer to such a cell controlled by a genome assembled from chemically synthesized pieces of DNA as a “synthetic cell,” even though the cytoplasm of the recipient cell is not synthetic. Phenotypic effects of the recipient cytoplasm are diluted with protein turnover and as cells carrying only the transplanted genome replicate. Following transplantation and replication on a plate to form a colony (>30 divisions or >109-fold dilution), progeny will not contain any protein molecules that were present in the original recipient cell (Lartigue et al., 2007).53 This was previously demonstrated when we first described genome transplantation (Lartigue et al., 2007). The properties of the cells controlled by the assembled genome are expected to be the same as if the whole cell had been produced synthetically (the DNA software builds its own hardware).
Ok. De fato, o DNA foi montado pelo homem. E foi inserido no citoplasma de uma célula viva, da qual foi extraído seu original DNA. A célula aceitou o DNA artificial e continuou sua vida normalmente, inclusive reproduzindo-se. Nas seguintes gerações não havia mais o citoplasma ou qualquer outro material do ser vivo. Tudo na nova geração era sintético.
Ok. Tambem a 4 bilhões de anos atras, a matéria dispersa e inorgânica deste planeta produziu moléculas organicas que nunca existiram aqui antes. Assim, o planeta foi como a célula que recebeu o DNA sintético, nunca existido nela antes. Para o planeta, as moléculas organicas seriam tambem “sintéticas”.
Mas no planeta isto aconteceu porque as informações para sistema natural e biológico estavam no ar e se infiltrando nos átomos terrestres. Estas informações seriam algo alienígena, sintético, nao natural. Mas mesmo assim, o planeta aceitou estas moléculas, elas foram desenvolvidas por aquelas informações alienígenas e formaram os seres vivos, que levam suas vidas normais, se reproduzem, etc.
Se a vida ja era sintetica em relacao a este planeta, e eles continuam no mesmo planeta, eles fizeram o que de sintetico?!
Para terminar o paper diz:
… the DNA software builds its own hardware.
Mas eu pensava que apenas eu tinha escrito e publicado esta frase a 30 anos atras, quando registrei os copyrights do manuscrito da Teoria da Matrix/DNA… E mesmo aqui neste website posso provar que antecipei este conceito. Este website foi publicado em 2008 com sua pagina HOME tal como esta até hoje, não foi mudada nem uma virgula ( mesmo porque para mudar uma virgula ali é preciso entender de linguagem HTML, dreamwiver, etc., coisas que não entendo, por isso não posso mexer ali). E o que foi registrado em 2008 na HOME?
4) A Matriz nos conduz a re-interpretar a Evolução Cosmológica e a Evolução Biológica erigindo uma nova versão da História Natural Universal. Também sugere que – assim como o homem é corpo e mente, o DNA é matéria e um comando desconhecido de instruções, e o computador é software mais hardware – o processo da evolução universal se compõe de hardware e software que interagem retroativamente entre si;
Mas este paper foi publicado 3 anos depois, em 2011, como se ve aqui:
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. capricolumrecipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The onlyDNA 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.
In 1977, Sanger and colleagues determined the complete genetic sequence of phage ϕX174 (Sanger et al., 1977), the first DNA genome to be completely sequenced. Eighteen years later, in 1995, our team was able to read the first complete genetic sequence of a self-replicating bacterium, Haemophilus influenzae (Fleischmann et al., 1995). Reading the genetic sequence of a wide range of species has increased exponentially from these early studies. The ability to rapidly digitize genomic information has increased by more than eight orders of magnitude over the past 25 years (Venter, 2010). Efforts to understand all this new genomic information have spawned numerous new computational and experimental paradigms, yet our genomic knowledge remains very limited. No single cellular system has all of its genes understood in terms of their biological roles. Even in simple bacterial cells, do the chromosomes contain the entire genetic repertoire? If so, can a complete genetic system be reproduced by chemical synthesis starting with only the digitized DNA sequence contained in a computer?
Our interest in synthesis of large DNA molecules and chromosomes grew out of our efforts over the past 15 years to build a minimal cell that contains only essential genes. This work was inaugurated in 1995 when we sequenced the genome of Mycoplasma genitalium, a bacterium with the smallest complement of genes of any known organism capable of independent growth in the laboratory. More than 100 of the 485 protein-coding genes of M. genitalium are dispensable when disrupted one at a time (Hutchison et al., 1999; Glass et al., 2006; Smith et al., 2008). We developed a strategy for assembling viral-sized pieces to produce large DNA molecules that enabled us to assemble a synthetic M. genitalium genome in four stages from chemically synthesized DNA cassettes averaging about 6 kb in size. This was
accomplished through a combination of in vitro enzymatic methods and in vivo recombination in Saccharomyces cerevisiae. The whole synthetic genome [582,970 base pairs (bp)] was stably grown as a yeast centromeric plasmid (YCp) (Gibson et al., 2008b).
Several hurdles were overcome in transplanting and expressing a chemically synthesized chromosome in a recipient cell. We needed to improve methods for extracting intact chromosomes from yeast. We also needed to learn how to transplant these genomes into a recipient bacterial cell to establish a cell controlled only by a synthetic genome. Because M. genitalium has an extremely slow growth rate, we turned to two faster-growing mycoplasma species, M. mycoidessubspecies capri (GM12) as donor, and M. capricolum subspecies capricolum (CK) as recipient.
To establish conditions and procedures for transplanting the synthetic genome out of yeast, we developed methods for cloning entire bacterial chromosomes as centromeric plasmids in yeast, including a native M. mycoides genome (Lartigue et al., 2009; Benders et al., 2010). However, initial attempts to extract the M. mycoides genome from yeast and transplant it into M. capricolum failed. We discovered that the donor and recipient mycoplasmas share a common restriction system. The donor genome was methylated in the native M. mycoides cells and was therefore protected against restriction during the transplantation from a native donor cell (Lartigue et al., 2007). However, the bacterial genomes grown in yeast are unmethylated and so are not protected from the single restriction system of the recipient cell. We overcame this restriction barrier by methylating the donor DNA with purified methylases or crude M. mycoidesor M. capricolum extracts, or by simply disrupting the recipient cell’s restriction system (Lartigue et al., 2009).
We now have combined all of our previously established procedures and report the synthesis, assembly, cloning, and successful transplantation of the 1.08-Mbp M. mycoides JCVI-syn1.0 genome, to create a new cell controlled by this synthetic genome.
Design of the M. mycoides JCVI-syn1.0 genome was based on the highly accurate finished genome sequences of two laboratory strains of M. mycoides subspecies capri GM12 (Lartigue et al., 2009; Benders et al., 2010).49 One was the genome donor used by Lartigue et al. [GenBank accession CP001621] (2007). The other was a strain created by transplantation of a genome that had been cloned and engineered in yeast, YCpMmyc1.1-ΔtypeIIIres [GenBank accession CP001668] (Lartigue et al., 2009). This project was critically dependent on the accuracy of these sequences. Although we believe that both finished M. mycoides genome sequences are reliable, there are 95 sites at which they differ. We began to design the synthetic genome before both sequences were finished. Consequently, most of the cassettes were designed and synthesized based on the CP001621 sequence.49 When it was finished, we chose the sequence of the genome successfully transplanted from yeast (CP001668) as our design reference (except that we kept the intact typeIIIres gene). All differences that appeared biologically significant between CP001668 and previously synthesized cassettes were corrected to match it exactly.49 Sequence differences between our synthetic cassettes andCP001668 that occurred at 19 sites appeared harmless and so were not corrected. These provide 19 polymorphic differences between our synthetic genome (JCVI-syn1.0) and the natural (nonsynthetic) genome (YCpMmyc1.1) that we have cloned in yeast and use as a standard for genome transplantation from yeast (Lartigue et al., 2009). To further differentiate between the syntheticgenome and the natural one, we designed four watermark sequences (fig. S1) to replace one or more cassettes in regions experimentally demonstrated [watermarks 1 (1246 bp) and 2 (1081 bp)] or predicted [watermarks 3 (1109 bp) and 4 (1222 bp)] to not interfere with cell viability. These watermark sequences encode unique identifiers while limiting their translation into peptides. Table S1 lists the differences between the synthetic genome and this natural standard. Figure S2 shows a map of the M. mycoides JCVI-syn1.0 genome. Cassette and assembly intermediate boundaries, watermarks, deletions, insertions, and genes of the M. mycoides JCVI syn1.0 are shown in fig. S2, and the sequence of the transplanted mycoplasma clone sMmYCp235-1 has been submitted to GenBank (accession CP002027).
The designed cassettes were generally 1080 bp with 80-bp overlaps to adjacent cassettes.50 They were all produced by assembly of chemically synthesized oligonucleotides by Blue Heron (Bothell, Washington). Each cassette was individually synthesized and sequence-verified by the manufacturer. To aid in the building process, DNA cassettes and assembly intermediates were designed to contain Not I restriction sites at their termini and recombined in the presence of vector elements to allow for growth and selection in yeast (Gibson et al., 2008b).50 A hierarchical strategy was designed to assemble the genome in three stages by transformation and homologous recombination in yeast from 1078 1-kb cassettes (Fig. A8-1) (Gibson, 2009; Gibson et al., 2008a)
In the first stage, cassettes and a vector were recombined in yeast and transferred to Escherichia coli.50 Plasmid DNAwas then isolated from individual E. coli clones and digested to screen for cells containing a vector with an assembled 10-kb insert. One successful 10-kb assembly is represented (Fig. A8-2A). In general, at least one 10-kb assembled fragment could be obtained by screening 10 yeast clones. However, the rate of success varied from 10 to 100%. All of the first-stage intermediates were sequenced. Nineteen out of 111 assemblies contained errors. Alternate clones were selected, sequence-verified, and moved on to the next assembly stage.
The pooled 10-kb assemblies and their respective cloning vectors were transformed into yeast as above to produce 100-kb assembly intermediates.50 Our results indicated that these products cannot be stably maintained in E. coli, so recombined DNA had to be extracted from yeast. Multiplex polymerase chain reaction (PCR) was performed on selected yeast clones (fig. S3 and table S2). Because every 10-kb assembly intermediate was represented by a primer pair in this analysis, the presence of all amplicons would suggest an assembled 100-kb intermediate. In general, 25% or more of the clones screened contained all of the amplicons expected for a complete assembly. One of these clones was selected for further screening. Circular plasmid DNA was extracted and sized on an agarose gel alongside a supercoiled marker. Successful second-stage assemblies with the vector sequence are ~105 kb in length (Fig. A8-2B). When all amplicons were produced following multiplex PCR, a second-stage assembly intermediate of the correct size was usually produced. In some cases, however, small deletions occurred. In other instances, multiple 10-kb fragments were assembled, which produced a larger second-stage assembly intermediate. Fortunately, these differences could easily be detected on an agarose gel before complete genome assembly.
In preparation for the final stage of assembly, it was necessary to isolate microgram quantities of each of the 11 second-stage assemblies.51 As reported (Devenish and Newlon, 1982), circular plasmids the size of our second-stage assemblies could be isolated from yeast spheroplasts after an alkaline-lysis procedure. To further purify the 11 assembly intermediates, they were treated with exonuclease and passed through an anion-exchange column. A small fraction of the total plasmid DNA (1/100) was digested with Not I and analyzed by field-inversion gel electrophoresis (FIGE) (Fig. A8-2C). This method produced ~1 mg of each assembly per 400 ml of yeast culture (~1011 cells).
The method above does not completely remove all of the linear yeast chromosomal DNA, which we found could substantially decrease the yeast transformation and assembly efficiency. To further enrich for the 11 circular assembly intermediates, ~200 ng samples of each assembly were pooled and mixed with molten agarose. As the agarose solidifies, the fibers thread through and topologically “trap” circular DNA (Dean et al., 1973). Untrapped linear DNA can then be separated out of the agarose plug by electrophoresis, thus enriching for the trapped circular molecules. The 11 circular assembly intermediates were digested with Not I so that the inserts could be released. Subsequently, the fragments were extracted from the agarose plug, analyzed by FIGE (Fig. A8-2D), and transformed into yeast spheroplasts.51 In this third and final stage of assembly, an additional vector sequence was not required because the yeast cloning elements were already present in assembly 811–900.
To screen for a complete genome, multiplex PCR was carried out with 11 primer pairs, designed to span each of the 11 100-kb assembly junctions (table S3). Of 48 colonies screened, DNA extracted from one clone (sMmYCp235) produced all 11 amplicons. PCR of the wild-type positive control (YCpMmyc1.1) produced an indistinguishable set of 11 amplicons (Fig. A8-3A). To further demonstrate the complete assembly of a synthetic M. mycoides genome, intact DNA was isolated from yeast in agarose plugs and subjected to two restriction analyses: Asc I and BssH II.52 Because these restriction sites are present in three of the four watermark sequences, this choice of digestion produces restriction patterns that are distinct from that of the natural M. mycoides genome (Figs. A8-1 and A8-3B). The sMmYCp235 clone produced the restriction pattern expected for a completely assembled synthetic genome (Fig. A8-3C).
Additional agarose plugs used in the gel analysis above (Fig. A8-3C) were also used in genome transplantation experiments.52 Intact synthetic M. mycoides genomes from the sMmYCp235 yeast clone were transplanted into restriction-minus M. capricolum recipient cells, as described (Lartigue et al., 2009). Results were scored by selecting for growth of blue colonies on SP4 medium containing tetracycline and X-gal at 37°C. Genomes isolated from this yeast clone produced 5 to 15 tetracycline-resistant blue colonies per agarose plug, a number comparable to that produced by the YCpMmyc1.1 control. Recovery of colonies in all transplantation experiments was dependent on the presence of both M. capricolum recipient cells and an M. mycoides genome.
To aid in testing the functionality of each 100-kb synthetic segment, semi-synthetic genomes were constructed and transplanted. By mixing natural pieces with synthetic ones, the successful construction of each synthetic 100-kb assembly could be verified without having to sequence these intermediates. We cloned 11 overlapping natural 100-kb assemblies in yeast by using a previously described method (Leem et al., 2003). In 11 parallel reactions, yeast cells were cotransformed with fragmented M. mycoides genomic DNA (YCpMmyc1.1) that averaged ~100 kb in length and aPCR-amplified vector designed to overlap the ends of the 100-kb inserts. To maintain the appropriate overlaps so that natural and synthetic fragments could be recombined, the PCR-amplified vectors were produced via primers with the same 40-bp overlaps used to clone the 100-kb synthetic assemblies. The semisynthetic genomes that were constructed contained between 2 and 10 of the 11 100-kb synthetic subassemblies (Table A8-1). The production of viable colonies produced after transplantation confirmed that the synthetic fraction of each genome contained no lethal mutations. Only one of the 100-kb subassemblies, 811–900, was not viable.
Initially, an error-containing 811–820 clone was used to produce a synthetic genome that did not transplant. This was expected because the error was a single– base pair deletion that creates a frameshift in dnaA, an essential gene for chromosomal replication. We were previously unaware of this mutation. By using a semisynthetic genome construction strategy, we pinpointed 811–900 as the source for failed synthetic transplantation experiments. Thus, we began to reassemble an error-free 811–900 assembly, which was used to produce the sMmYCp235 yeast strain. The dnaA-mutated genome differs by only one nucleotide from the synthetic genome in sMmYCp235. This genome served as a negative control in our transplantation experiments. The dnaA mutation was also repaired at the 811–900 level by genome engineering in yeast (Noskov et al., 2010). A repaired 811–900 assembly was used in a final-stage assembly to produce a yeast clone with a repaired genome. This yeast clone is named sMmYCP142 and could be transplanted. A complete list of genomes that have been assembled from 11 pieces and successfully transplanted is provided in Table A8-1.
To rapidly distinguish the synthetic transplants from M. capricolum or natural M. mycoides, two analyses were performed. First, four primer pairs that are specific to each of the four watermarks were designed such that they produce four amplicons in a single multiplex PCR reaction (table S4). All four amplicons were produced by transplants generated from sMmYCp235, but not YCpMmyc1.1 (Fig. A8-4A). Second, the gel analysis with Asc I and BssH II, described above (Fig. A8-3C), was performed. The restriction pattern obtained was consistent with a transplant produced from a synthetic M. mycoides genome (Fig. A8-4B).
A single transplant originating from the sMmYCp235 synthetic genome was sequenced. We refer to this strain as M. mycoides JCVIsyn1.0. The sequence matched the intended design with the exception of the known polymorphisms, eight new single-nucleotide polymorphisms, an E. coli transposon insertion, and an 85-bp duplication (table S1). The transposon insertion exactly matches the size and sequence of IS1, a transposon in E. coli. It is likely that IS1 infected the 10-kb subassembly following its transfer to E. coli. The IS1 insert is flanked by direct repeats of M. mycoidessequence, suggesting that it was inserted by a transposition mechanism. The 85-bp duplication is a result of a nonhomologous end joining event, which was not detected in our sequence analysis at the 10-kb stage. These two insertions disrupt two genes that are evidently nonessential. We did not find any sequences in the synthetic genome that could be identified as belonging to M. capricolum. This indicates that there was a complete replacement of the M. capricolum genome by our synthetic genome during the trans-plant process. The cells with only the synthetic genome are self-replicating and capable of logarithmic growth. Scanning and transmission electron micrographs (EMs) of M. mycoides JCVI-syn1.0 cells show small, ovoid cells surrounded by cytoplasmic membranes (Fig. A8-5, C to F). Proteomic analysis of M. mycoides JCVI-syn1.0 and the wild-type control (YCpMmyc1.1) by two-dimensional gel electrophoresis revealed almost identical patterns of protein spots (fig. S4) that differed from those previously reported for M. capricolum (Lartigue et al., 2007). Fourteen genes are deleted or disrupted in the M. mycoides JCVI-syn1.0 genome; however, the rate of appearance of colonies on agar plates and the colony morphology are similar (compareFig. A8-5, A and B). We did observe slight differences in the growth rates in a color-changing unit assay, with the JCVI-syn1.0 transplants growing slightly faster than the MmcyYCp1.1 control strain (fig. S6)
In 1995, the quality standard for sequencing was considered to be one error in 10,000 bp, and the sequencing of a microbial genome required months. Today, the accuracy is substantially higher. Genome coverage of 30 to 50× is not unusual, and sequencing only requires a few days. However, obtaining an error-free genome that could be transplanted into a recipient cell to create a new cell controlled only by the synthetic genome was complicated and required many quality-control steps. Our success was thwarted for many weeks by a single–base pair deletion in the essential genednaA. One wrong base out of more than 1 million in an essential gene rendered the genome inactive, whereas major genome insertions and deletions in nonessential parts of the genome had no observable effect on viability. The demonstration that our synthetic genome gives rise to transplants with the characteristics of M. mycoides cells implies that the DNA sequence on which it is based is accurate enough to specify a living cell with the appropriate properties.
Our synthetic genomic approach stands in sharp contrast to various other approaches to genome engineering that modify natural genomes by introducing multiple insertions, substitutions, or deletions (Itaya et al., 2005; Itaya, 1995; Mizoguchi et al., 2007; Chun et al., 2007; Wang et al., 2009). This work provides a proof of principle for producing cells based on computer-designed genome sequences. DNA sequencing of a cellular genome allows storage of the genetic instructions for life as a digital file.
The synthetic genome described here has only limited modifications from the naturally occurring M. mycoides genome. However, the approach we have developed should be applicable to the synthesis and transplantation of more novel genomes as genome design progresses (Khalil and Collins, 2010). We refer to such a cell controlled by a genome assembled from chemically synthesized pieces of DNA as a “synthetic cell,” even though the cytoplasm of the recipient cell is not synthetic. Phenotypic effects of the recipient cytoplasm are diluted with protein turnover and as cells carrying only the transplanted genome replicate. Following transplantation and replication on a plate to form a colony (>30 divisions or >109-fold dilution), progeny will not contain any protein molecules that were present in the original recipient cell (Lartigue et al., 2007).53 This was previously demonstrated when we first described genome transplantation (Lartigue et al., 2007). The properties of the cells controlled by the assembled genome are expected to be the same as if the whole cell had been produced synthetically (the DNA software builds its own hardware).
The ability to produce synthetic cells renders it essential for researchers making synthetic DNA constructs and cells to clearly watermark their work to distinguish it from naturally occurring DNA and cells. We have watermarked the synthetic chromosome in this and our previous study (Gibson, et al., 2008b).
If the methods described here can be generalized, design, synthesis, assembly, and transplantation of synthetic chromosomes will no longer be a barrier to the progress of synthetic biology. We expect that the cost of DNA synthesiswill follow what has happened with DNA sequencing and continue to exponentially decrease. Lower synthesis costs combined with automation will enable broad applications for synthetic genomics.
We have been driving the ethical discussion concerning synthetic life from the earliest stages of this work (Cho et al., 1999; Garfinkel et al., 2007). As synthetic genomic applications expand, we anticipate that this work will continue to raise philosophical issues that have broad societal and ethical implications. We encourage the continued discourse
Ver References no artigo.
Trata-se de um pacote de videos/aulas intitulado “Origin of Life and Molecular Evolution”. Sendo uma abordagem academica atualizada, ela difere da abordagem e interpretacoes da minha Matrix/DNA Theory. Vou aqui ver os principais videos (comecando pelo segundo, do Jack Szostak) e registrar os pontos em conflito, analizando-os.
Primeiro video visto:
Jack esta aqui pregando uma teoria da origem da vida. Se essa teoria estiver errada, ou muito incompleta, ainda não sera esta geração de jovens e estudantes que irão eliminar as grandes doenças mortais, tradicionais, pois a geração de Jack que acreditou e obedeceu a esta teoria, não o fez. E isto e’ tao grave que algum querido seu, ou ate’ mesmo você, venha também a ser torturado ate a morte… por causa de uma teoria errada.
Primeiro estou copiando aqui meus comentários postado no debate no Youtube. A seguir, estou analisando os principais pontos focados no video.
Meus comentários postado no Youtube:
Louis Charles Morelli – Out(10)/25/2016