RESUMO
Prodigiosin, a red linear tripyrrole pigment and a member of the prodiginine family, is normally secreted by the human pathogen Serratia marcescens as a secondary metabolite. Studies on prodigiosin have received renewed attention as a result of reported immunosuppressive, antimicrobial and anticancer properties. High-level synthesis of prodigiosin and the bioengineering of strains to synthesise useful prodiginine derivatives have also been a subject of investigation. To exploit the potential use of prodigiosin as a clinical drug targeting bacteria or as a dye for textiles, high-level synthesis of prodigiosin is a prerequisite. This review presents an overview on the biosynthesis of prodigiosin from its natural host Serratia marcescens and through recombinant approaches as well as highlighting the beneficial properties of prodigiosin. We also discuss the prospect of adopting a synthetic biology approach for safe and cost-effective production of prodigiosin in a more industrially compliant surrogate host.
Assuntos
Antibacterianos/metabolismo , Antineoplásicos/metabolismo , Imunossupressores/metabolismo , Pigmentos Biológicos/metabolismo , Prodigiosina/metabolismo , Serratia marcescens/metabolismo , Vias Biossintéticas/genética , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Serratia marcescens/genética , Biologia Sintética/métodosRESUMO
BACKGROUND: Violacein is a deep violet compound that is produced by a number of bacterial species. It is synthesized from tryptophan by a pathway that involves the sequential action of 5 different enzymes (encoded by genes vioA to vioE). Violacein has antibacterial, antiparasitic, and antiviral activities, and also has the potential of inducing apoptosis in certain cancer cells. RESULTS: Here, we describe the construction of a series of plasmids harboring the complete or partial violacein biosynthesis operon and their use to enable production of violacein and deoxyviolacein in E.coli. We performed in vitro assays to determine the biological activity of these compounds against Plasmodium, Trypanosoma, and mammalian cells. We found that, while deoxyviolacein has a lower activity against parasites than violacein, its toxicity to mammalian cells is insignificant compared to that of violacein. CONCLUSIONS: We constructed E. coli strains capable of producing biologically active violacein and related compounds, and propose that deoxyviolacein might be a useful starting compound for the development of antiparasite drugs.
Assuntos
Antimaláricos/farmacologia , Antineoplásicos/farmacologia , Alcaloides Indólicos/farmacologia , Indóis/farmacologia , Compostos de Espiro/farmacologia , Tripanossomicidas/farmacologia , Animais , Antimaláricos/isolamento & purificação , Antimaláricos/metabolismo , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Células COS , Chlorocebus aethiops , Escherichia coli/genética , Células Hep G2 , Humanos , Alcaloides Indólicos/isolamento & purificação , Alcaloides Indólicos/metabolismo , Indóis/isolamento & purificação , Indóis/metabolismo , Engenharia Metabólica , Óperon , Plasmídeos/genética , Plasmodium falciparum/efeitos dos fármacos , Compostos de Espiro/isolamento & purificação , Compostos de Espiro/metabolismo , Tripanossomicidas/isolamento & purificação , Tripanossomicidas/metabolismo , Trypanosoma cruzi/efeitos dos fármacosRESUMO
BACKGROUND: Correlation matrices are important in inferring relationships and networks between regulatory or signalling elements in biological systems. With currently available technology sample sizes for experiments are typically small, meaning that these correlations can be difficult to estimate. At a genome-wide scale estimation of correlation matrices can also be computationally demanding. RESULTS: We develop an empirical Bayes approach to improve covariance estimates for gene expression, where we assume the covariance matrix takes a block diagonal form. Our method shows lower false discovery rates than existing methods on simulated data. Applied to a real data set from Bacillus subtilis we demonstrate it's ability to detecting known regulatory units and interactions between them. CONCLUSIONS: We demonstrate that, compared to existing methods, our method is able to find significant covariances and also to control false discovery rates, even when the sample size is small (n=10). The method can be used to find potential regulatory networks, and it may also be used as a pre-processing step for methods that calculate, for example, partial correlations, so enabling the inference of the causal and hierarchical structure of the networks.
Assuntos
Bacillus subtilis/genética , Teorema de Bayes , Simulação por Computador , Tamanho da Amostra , Software , Transcrição GênicaRESUMO
DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Edição de Genes/métodos , Engenharia Genética/métodos , Recombinação Genética/genética , Biologia Sintética/métodos , Genoma Bacteriano/genética , Peso MolecularRESUMO
BACKGROUND: Escherichia coli K-12 is a frequently used host for a number of synthetic biology and biotechnology applications and chassis for the development of the minimal cell factories. Novel approaches for integrating high molecular weight DNA into the E. coli chromosome would therefore greatly facilitate engineering efforts in this bacterium. RESULTS: We developed a reliable and flexible lambda Red recombinase-based system, which utilizes overlapping DNA fragments for integration of the high molecular weight DNA into the E. coli chromosome. Our chromosomal integration strategy can be used to integrate high molecular weight DNA of variable length into any non-essential locus in the E. coli chromosome. Using this approach we integrated 15 kb DNA encoding sucrose catabolism and lactose metabolism and transport operons into the fliK locus of the flagellar region 3b in the E. coli K12 MG1655 chromosome. Furthermore, with this system we integrated 50 kb of Bacillus subtilis 168 DNA into two target sites in the E. coli K12 MG1655 chromosome. The chromosomal integrations into the fliK locus occurred with high efficiency, inhibited motility, and did not have a negative effect on the growth of E. coli. CONCLUSIONS: In addition to the rational design of synthetic biology devices, our high molecular weight DNA chromosomal integration system will facilitate metabolic and genome-scale engineering of E. coli.
Assuntos
Cromossomos Bacterianos/genética , Escherichia coli K12/genética , Recombinases/metabolismo , Recombinação Genética , Bacillus subtilis/genética , DNA Bacteriano/genética , Escherichia coli K12/crescimento & desenvolvimento , Escherichia coli K12/metabolismo , Lactose/metabolismo , Peso Molecular , Recombinases/genética , Sacarose/metabolismo , Biologia Sintética/métodosRESUMO
Marked phenotypic variation characterizes isolates of Toxoplasma gondii, a ubiquitous zoonotic parasite that serves as an important experimental model for studying apicomplexan parasites. Progress in identifying the heritable basis for clinically and epidemiologically significant differences requires a robust system for describing and interpreting evolutionary subdivisions in this prevalent pathogen. To develop such a system, we have examined more than 950 isolates collected from around the world and genotyped them using three independent sets of polymorphic DNA markers, sampling 30 loci distributed across all nuclear chromosomes as well as the plastid genome. Our studies reveal a biphasic pattern consisting of regions in the Northern Hemisphere where a few, highly clonal and abundant lineages predominate; elsewhere, and especially in portions of South America are characterized by a diverse assemblage of less common genotypes that show greater evidence of recombination. Clustering methods were used to organize the marked genetic diversity of 138 unique genotypes into 15 haplogroups that collectively define six major clades. Analysis of gene flow indicates that a small number of ancestral lineages gave rise to the existing diversity through a process of limited admixture. Identification of reference strains for these major groups should facilitate future studies on comparative genomics and identification of genes that control important biological phenotypes including pathogenesis and transmission.
Assuntos
Variação Genética , Filogenia , Toxoplasma/classificação , Toxoplasma/isolamento & purificação , Sequência de Bases , Loci Gênicos/genética , Marcadores Genéticos , Genética Populacional , Geografia , Haplótipos/genética , Íntrons/genética , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Dinâmica Populacional , Toxoplasma/genéticaRESUMO
BACKGROUND: Toxoplasma gondii is a widespread protozoan parasite of animals that causes zoonotic disease in humans. Three clonal variants predominate in North America and Europe, while South American strains are genetically diverse, and undergo more frequent recombination. All three northern clonal variants share a monomorphic version of chromosome Ia (ChrIa), which is also found in unrelated, but successful southern lineages. Although this pattern could reflect a selective advantage, it might also arise from non-Mendelian segregation during meiosis. To understand the inheritance of ChrIa, we performed a genetic cross between the northern clonal type 2 ME49 strain and a divergent southern type 10 strain called VAND, which harbors a divergent ChrIa. RESULTS: NextGen sequencing of haploid F1 progeny was used to generate a genetic map revealing a low level of conventional recombination, with an unexpectedly high frequency of short, double crossovers. Notably, both the monomorphic and divergent versions of ChrIa were isolated with equal frequency. As well, ChrIa showed no evidence of being a sex chromosome, of harboring an inversion, or distorting patterns of segregation. Although VAND was unable to self fertilize in the cat, it underwent successful out-crossing with ME49 and hybrid survival was strongly associated with inheritance of ChrIII from ME49 and ChrIb from VAND. CONCLUSIONS: Our findings suggest that the successful spread of the monomorphic ChrIa in the wild has not been driven by meiotic drive or related processes, but rather is due to a fitness advantage. As well, the high frequency of short double crossovers is expected to greatly increase genetic diversity among progeny from genetic crosses, thereby providing an unexpected and likely important source of diversity.
Assuntos
Troca Genética , Variação Genética , Toxoplasma/genética , Animais , Gatos , Mapeamento Cromossômico , Cromossomos , Cruzamentos Genéticos , Evolução Molecular , Ligação Genética , Genoma de Protozoário , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Polimorfismo de Nucleotídeo Único , Recombinação GenéticaRESUMO
While synthetic biology is hoped to hold promise and potential to address pressing global challenges, the issue of regulation is an under-appreciated challenge. Particularly in Europe, the regulatory frameworks involved are rooted in historical concepts based on containment and release. Through a series of case studies including a field-use biosensor intended to detect arsenic in well water in Nepal and Bangladesh, and insects engineered for sterility, we explore the implications that this regulatory and conceptual divide has had on the deployment of synthetic biology projects in different national contexts. We then consider some of the broader impacts that regulation can have on the development of synthetic biology as a field, not only in Europe but also globally, with a particular emphasis on low- and middle-income countries. We propose that future regulatory adaptability would be increased by moving away from a containment and release dichotomy and toward a more comprehensive assessment that accounts for the possibility of varying degrees of 'contained release'. Graphical Abstract.
RESUMO
Pathogenicity differences among laboratory isolates of the dominant clonal North American and European lineages of Toxoplasma gondii are largely controlled by polymorphisms and expression differences in rhoptry secretory proteins (ROPs). However, the extent to which such differences control virulence in natural isolates of T. gondii, including those from more diverse genetic backgrounds, is uncertain. We elucidated the evolutionary history and functional consequences of diversification in the serine/threonine kinase ROP18, a major virulence determinant in the mouse model. We characterized the extent of sequence polymorphism and the evolutionary forces acting on ROP18 and several antigen-encoding genes within a large collection of natural isolates, comparing them to housekeeping genes and introns. Surprisingly, despite substantial genetic diversity between lineages, we identified just three principal alleles of ROP18, which had very ancient ancestry compared to other sampled loci. Expression and allelic differences between these three alleles of ROP18 accounted for much of the variation in acute mouse virulence among natural isolates. While the avirulent type III allele was the most ancient, intermediate virulent (type II) and highly virulent (type I) lineages predominated and showed evidence of strong selective pressure. Out-group comparison indicated that historical loss of an upstream regulatory element increased ROP18 expression, exposing it to newfound diversifying selection, resulting in greatly enhanced virulence in the mouse model and expansion of new lineages. Population sweeps are evident in many genomes, yet their causes and evolutionary histories are rarely known. Our results establish that up-regulation of expression and selection at ROP18 in T. gondii has resulted in three distinct alleles with widely different levels of acute virulence in the mouse model. Preservation of all three alleles in the wild indicates they are likely adaptations for different niches. Our findings demonstrate that sweeping changes in population structure can result from alterations in a single gene.
Assuntos
Proteínas Serina-Treonina Quinases/genética , Proteínas de Protozoários/genética , Seleção Genética , Toxoplasma/genética , Toxoplasma/patogenicidade , Toxoplasmose/parasitologia , Alelos , Animais , Evolução Molecular , Variação Genética , Camundongos , Filogenia , Polimorfismo Genético , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/classificação , Toxoplasma/enzimologia , Regulação para Cima , VirulênciaRESUMO
Mechanisms of gene regulation are poorly understood in Apicomplexa, a phylum that encompasses deadly human pathogens like Plasmodium and Toxoplasma. Initial studies suggest that epigenetic phenomena, including histone modifications and chromatin remodeling, have a profound effect upon gene expression and expression of virulence traits. Using the model organism Toxoplasma gondii, we characterized the epigenetic organization and transcription patterns of a contiguous 1% of the T. gondii genome using custom oligonucleotide microarrays. We show that methylation and acetylation of histones H3 and H4 are landmarks of active promoters in T. gondii that allow us to deduce the position and directionality of gene promoters with >95% accuracy. These histone methylation and acetylation "activation" marks are strongly associated with gene expression. We also demonstrate that the pattern of histone H3 arginine methylation distinguishes certain promoters, illustrating the complexity of the histone modification machinery in Toxoplasma. By integrating epigenetic data, gene prediction analysis, and gene expression data from the tachyzoite stage, we illustrate feasibility of creating an epigenomic map of T. gondii tachyzoite gene expression. Further, we illustrate the utility of the epigenomic map to empirically and biologically annotate the genome and show that this approach enables identification of previously unknown genes. Thus, our epigenomics approach provides novel insights into regulation of gene expression in the Apicomplexa. In addition, with its compact genome, genetic tractability, and discrete life cycle stages, T. gondii provides an important new model to study the evolutionarily conserved components of the histone code.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas/genética , Toxoplasma/genética , Alquilação , Animais , Genoma , Histonas/metabolismo , Metilação , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/crescimento & desenvolvimentoRESUMO
BACKGROUND: Worldwide, approximately two billion people are chronically infected with Toxoplasma gondii with largely unknown consequences. METHODS: To better understand long-term effects and pathogenesis of this common, persistent brain infection, mice were infected at a time in human years equivalent to early to mid adulthood and studied 5-12 months later. Appearance, behavior, neurologic function and brain MRIs were studied. Additional analyses of pathogenesis included: correlation of brain weight and neurologic findings; histopathology focusing on brain regions; full genome microarrays; immunohistochemistry characterizing inflammatory cells; determination of presence of tachyzoites and bradyzoites; electron microscopy; and study of markers of inflammation in serum. Histopathology in genetically resistant mice and cytokine and NRAMP knockout mice, effects of inoculation of isolated parasites, and treatment with sulfadiazine or alphaPD1 ligand were studied. RESULTS: Twelve months after infection, a time equivalent to middle to early elderly ages, mice had behavioral and neurological deficits, and brain MRIs showed mild to moderate ventricular dilatation. Lower brain weight correlated with greater magnitude of neurologic abnormalities and inflammation. Full genome microarrays of brains reflected inflammation causing neuronal damage (Gfap), effects on host cell protein processing (ubiquitin ligase), synapse remodeling (Complement 1q), and also increased expression of PD-1L (a ligand that allows persistent LCMV brain infection) and CD 36 (a fatty acid translocase and oxidized LDL receptor that mediates innate immune response to beta amyloid which is associated with pro-inflammation in Alzheimer's disease). Immunostaining detected no inflammation around intra-neuronal cysts, practically no free tachyzoites, and only rare bradyzoites. Nonetheless, there were perivascular, leptomeningeal inflammatory cells, particularly contiguous to the aqueduct of Sylvius and hippocampus, CD4+ and CD8+ T cells, and activated microglia in perivascular areas and brain parenchyma. Genetically resistant, chronically infected mice had substantially less inflammation. CONCLUSION: In outbred mice, chronic, adult acquired T. gondii infection causes neurologic and behavioral abnormalities secondary to inflammation and loss of brain parenchyma. Perivascular inflammation is prominent particularly contiguous to the aqueduct of Sylvius and hippocampus. Even resistant mice have perivascular inflammation. This mouse model of chronic T. gondii infection raises questions of whether persistence of this parasite in brain can cause inflammation or neurodegeneration in genetically susceptible hosts.
Assuntos
Encéfalo/parasitologia , Encefalite/parasitologia , Degeneração Neural/parasitologia , Neurônios/parasitologia , Toxoplasmose Cerebral/fisiopatologia , Fatores Etários , Animais , Atrofia/parasitologia , Atrofia/patologia , Atrofia/fisiopatologia , Comportamento Animal/fisiologia , Biomarcadores/análise , Biomarcadores/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/parasitologia , Doença Crônica , Modelos Animais de Doenças , Encefalite/patologia , Encefalite/fisiopatologia , Feminino , Ventrículos Laterais/patologia , Imageamento por Ressonância Magnética , Camundongos , Microglia/imunologia , Microglia/parasitologia , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/patologia , Toxoplasma/citologia , Toxoplasma/fisiologia , Toxoplasmose Cerebral/patologiaRESUMO
Toxoplasma gondii is a highly successful protozoan parasite in the phylum Apicomplexa, which contains numerous animal and human pathogens. T.gondii is amenable to cellular, biochemical, molecular and genetic studies, making it a model for the biology of this important group of parasites. To facilitate forward genetic analysis, we have developed a high-resolution genetic linkage map for T.gondii. The genetic map was used to assemble the scaffolds from a 10X shotgun whole genome sequence, thus defining 14 chromosomes with markers spaced at approximately 300 kb intervals across the genome. Fourteen chromosomes were identified comprising a total genetic size of approximately 592 cM and an average map unit of approximately 104 kb/cM. Analysis of the genetic parameters in T.gondii revealed a high frequency of closely adjacent, apparent double crossover events that may represent gene conversions. In addition, we detected large regions of genetic homogeneity among the archetypal clonal lineages, reflecting the relatively few genetic outbreeding events that have occurred since their recent origin. Despite these unusual features, linkage analysis proved to be effective in mapping the loci determining several drug resistances. The resulting genome map provides a framework for analysis of complex traits such as virulence and transmission, and for comparative population genetic studies.
Assuntos
Genoma de Protozoário , Recombinação Genética , Toxoplasma/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos , Cruzamentos Genéticos , Bases de Dados de Ácidos Nucleicos , Resistência a Medicamentos/genética , Etiquetas de Sequências Expressas , Ligação Genética , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , Toxoplasma/efeitos dos fármacosRESUMO
The majority of the good DNA editing techniques have been developed in Escherichia coli; however, Bacillus subtilis is better host for a plethora of synthetic biology and biotechnology applications. Reliable and efficient systems for the transfer of synthetic DNA between E. coli and B. subtilis are therefore of the highest importance. Using synthetic biology approaches, such as streamlined lambda Red recombineering and Gibson Isothermal Assembly, we integrated genetic circuits pT7L123, Repr-ts-1 and pLT7pol encoding the lysis genes of bacteriophages MS2, ΦX174 and lambda, the thermosensitive repressor and the T7 RNA polymerase into the E. coli chromosome. In this system, T7 RNA polymerase regulated by the thermosensitive repressor drives the expression of the phage lysis genes. We showed that T7 RNA polymerase significantly increases efficiency of cell lysis and transfer of the plasmid and bacterial artificial chromosome-encoded DNA from the lysed E. coli into B. subtilis. The T7 RNA polymerase-driven inducible cell lysis system is suitable for the efficient cell lysis and transfer of the DNA engineered in E. coli to other naturally competent hosts, such as B. subtilis.
Assuntos
Bacillus subtilis/genética , Bacteriófagos/enzimologia , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Proteínas Virais/metabolismo , Bacillus subtilis/metabolismo , Competência de Transformação por DNA , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Biologia Sintética , Proteínas Virais/genéticaRESUMO
Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis.
Assuntos
Bacillus subtilis/genética , Cromossomos Artificiais Bacterianos/genética , Cromossomos Bacterianos , Engenharia Genética/métodos , Recombinases Rec A/genética , Recombinação Genética , Escherichia coli/genética , Plasmídeos , Biologia Sintética/métodos , Transformação BacterianaRESUMO
The ability to efficiently and reliably transfer genetic circuits between the key synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the major hurdles of the rational genome engineering. Using lambda Red recombineering we integrated the thermosensitive lambda repressor and the lysis genes of several bacteriophages into the E. coli chromosome. The lysis of the engineered autolytic cells is inducible by a simple temperature shift. We improved the lysis efficiency by introducing different combinations of lysis genes from bacteriophages lambda, ΦX174 and MS2 under the control of the thermosensitive lambda repressor into the E. coli chromosome. We tested the engineered autolytic cells by transferring plasmid and bacterial artificial chromosome (BAC)-borne genetic circuits from E. coli to B. subtilis. Our engineered system combines benefits of the two main synthetic biology chassis, E. coli and B. subtilis, and allows reliable and efficient transfer of DNA edited in E. coli into B. subtilis.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/virologia , Bacteriólise/genética , Bacteriófagos/fisiologia , Escherichia coli/genética , Escherichia coli/virologia , Transdução Genética , Plasmídeos/genéticaRESUMO
Accurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators. However, transcription rates observed from promoters can vary significantly depending on the growth rate of host cells and the experimental and genetic contexts of the measurement. Furthermore, in vivo measurement methods must accommodate variation in translation, protein folding, and maturation rates of reporter proteins, as well as metabolic load. The external factors affecting transcription activity may be considered to be extrinsic, and the goal of characterization should be to obtain quantitative measures of the intrinsic characteristics of promoters. We have developed a promoter characterization method that is based on a mathematical model for cell growth and reporter gene expression and exploits multiple in vivo measurements to compensate for variation due to extrinsic factors. First, we used optical density and fluorescent reporter gene measurements to account for the effect of differing cell growth rates. Second, we compared the output of reporter genes to that of a control promoter using concurrent dual-channel fluorescence measurements. This allowed us to derive a quantitative promoter characteristic (ρ) that provides a robust measure of the intrinsic properties of a promoter, relative to the control. We imposed different extrinsic factors on growing cells, altering carbon source and adding bacteriostatic agents, and demonstrated that the use of ρ values reduced the fraction of variance due to extrinsic factors from 78% to less than 4%. This is a simple and reliable method to quantitatively describe promoter properties.
Assuntos
Regiões Promotoras Genéticas , Fluorescência , Genes Reporter , Proteínas Luminescentes/metabolismo , Plasmídeos/metabolismo , Biossíntese de ProteínasRESUMO
Toxoplasma gondii is among the most prevalent parasites worldwide, infecting many wild and domestic animals and causing zoonotic infections in humans. T. gondii differs substantially in its broad distribution from closely related parasites that typically have narrow, specialized host ranges. To elucidate the genetic basis for these differences, we compared the genomes of 62 globally distributed T. gondii isolates to several closely related coccidian parasites. Our findings reveal that tandem amplification and diversification of secretory pathogenesis determinants is the primary feature that distinguishes the closely related genomes of these biologically diverse parasites. We further show that the unusual population structure of T. gondii is characterized by clade-specific inheritance of large conserved haploblocks that are significantly enriched in tandemly clustered secretory pathogenesis determinants. The shared inheritance of these conserved haploblocks, which show a different ancestry than the genome as a whole, may thus influence transmission, host range and pathogenicity.
Assuntos
Genoma de Protozoário , Toxoplasma/genética , Toxoplasma/patogenicidade , Sequência Conservada , DNA de Protozoário/genética , Regulação da Expressão Gênica/fisiologia , Filogenia , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sintenia , VirulênciaRESUMO
BACKGROUND: A common feature of microarray experiments is the occurrence of missing gene expression data. These missing values occur for a variety of reasons, in particular, because of the filtering of poor quality spots and the removal of undefined values when a logarithmic transformation is applied to negative background-corrected intensities. The efficiency and power of an analysis performed can be substantially reduced by having an incomplete matrix of gene intensities. Additionally, most statistical methods require a complete intensity matrix. Furthermore, biases may be introduced into analyses through missing information on some genes. Thus methods for appropriately replacing (imputing) missing data and/or weighting poor quality spots are required. RESULTS: We present a likelihood-based method for imputing missing data or weighting poor quality spots that requires a number of biological or technical replicates. This likelihood-based approach assumes that the data for a given spot arising from each channel of a two-dye (two-channel) cDNA microarray comparison experiment independently come from a three-component mixture distribution--the parameters of which are estimated through use of a constrained E-M algorithm. Posterior probabilities of belonging to each component of the mixture distributions are calculated and used to decide whether imputation is required. These posterior probabilities may also be used to construct quality weights that can down-weight poor quality spots in any analysis performed afterwards. The approach is illustrated using data obtained from an experiment to observe gene expression changes with 24 hr paclitaxel (Taxol) treatment on a human cervical cancer derived cell line (HeLa). CONCLUSION: As the quality of microarray experiments affect downstream processes, it is important to have a reliable and automatic method of identifying poor quality spots and arrays. We propose a method of identifying poor quality spots, and suggest a method of repairing the arrays by either imputation or assigning quality weights to the spots. This repaired data set would be less biased and can be analysed using any of the appropriate statistical methods found in the microarray literature.
Assuntos
Algoritmos , Funções Verossimilhança , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias do Colo do Útero/genética , Feminino , Células HeLa , Humanos , Modelos Estatísticos , Controle de QualidadeRESUMO
Escherichia coli is used as a chassis for a number of Synthetic Biology applications. The lack of suitable chromosomal integration and expression loci is among the main hurdles of the E. coli engineering efforts. We identified and validated chromosomal integration and expression target sites within E. coli K12 MG1655 flagellar region 1. We analyzed five open reading frames of the flagellar region 1, flgA, flgF, flgG, flgI, and flgJ, that are well-conserved among commonly-used E. coli strains, such as MG1655, W3110, DH10B and BL21-DE3. The efficiency of the integration into the E. coli chromosome and the expression of the introduced genetic circuit at the investigated loci varied significantly. The integrations did not have a negative impact on growth; however, they completely abolished motility. From the investigated E. coli K12 MG1655 flagellar region 1, flgA and flgG are the most suitable chromosomal integration and expression loci.
Assuntos
Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , Escherichia coli/genética , Loci Gênicos , Recombinação Genética , Fases de Leitura AbertaRESUMO
The Gram-negative bacterium Escherichia coli is routinely used as the chassis for a variety of biotechnology and synthetic biology applications. Identification and analysis of reliable chromosomal integration and expression target loci is crucial for E. coli engineering. Chromosomal loci differ significantly in their ability to support integration and expression of the integrated genetic circuits. In this study, we investigate E. coli K12 MG1655 flagellar regions 2 and 3b. Integration of the genetic circuit into seven and nine highly conserved genes of the flagellar regions 2 (motA, motB, flhD, flhE, cheW, cheY and cheZ) and 3b (fliE, F, G, J, K, L, M, P, R), respectively, showed significant variation in their ability to support chromosomal integration and expression of the integrated genetic circuit. While not reducing the growth of the engineered strains, the integrations into all 16 target sites led to the loss of motility. In addition to high expression, the flagellar region 3b supports the highest efficiency of integration of all E. coliâ K12 MG1655 flagellar regions and is therefore potentially the most suitable for the integration of synthetic genetic circuits.