Article added to library!
x
Pubchase is a service of protocols.io - free, open access, crowdsourced protocols repository. Explore protocols.
Sign in
Reset password
or connect with
Facebook
By signing in you are agreeing to our
Terms Of Service and Privacy Policy
Synthetic Biology
SMRT Gate: A method for validation of synthetic constructs on Pacific Biosciences sequencing platforms
Jul 13, 2017   BioTechniques
D'Amore R, Johnson J, Haldenby S, Hall N, Hughes M, Joynson R, Kenny JG, Patron N, Hertz-Fowler C, Hall A
SMRT Gate: A method for validation of synthetic constructs on Pacific Biosciences sequencing platforms
Jul 13, 2017
BioTechniques
Current DNA assembly methods are prone to sequence errors, requiring rigorous quality control (QC) to identify incorrect assemblies or synthesized constructs. Such errors can lead to misinterpretation of phenotypes. Because of this intrinsic problem, routine QC analysis is generally performed on three or more clones using a combination of restriction endonuclease assays, colony PCR, and Sanger sequencing. However, as new automation methods emerge that enable high-throughput assembly, QC using these techniques has become a major bottleneck. Here, we describe a quick and affordable methodology for the QC of synthetic constructs. Our method involves a one-pot digestion-ligation DNA assembly reaction, based on the Golden Gate assembly methodology, that is coupled with Pacific Biosciences' Single Molecule, Real-Time (PacBio SMRT) sequencing technology.
Computational design of a symmetrical β-trefoil lectin with cancer cell binding activity
Jul 20, 2017   Scientific Reports
Terada D, Voet ARD, Noguchi H, Kamata K, Ohki M, Addy C, Fujii Y, Yamamoto D, Ozeki Y, Tame JRH, Zhang KYJ
Computational design of a symmetrical β-trefoil lectin with cancer cell binding activity
Jul 20, 2017
Scientific Reports
Computational protein design has advanced very rapidly over the last decade, but there remain few examples of artificial proteins with direct medical applications. This study describes a new artificial β-trefoil lectin that recognises Burkitt's lymphoma cells, and which was designed with the intention of finding a basis for novel cancer treatments or diagnostics. The new protein, called "Mitsuba", is based on the structure of the natural shellfish lectin MytiLec-1, a member of a small lectin family that uses unique sequence motifs to bind α-D-galactose. The three subdomains of MytiLec-1 each carry one galactose binding site, and the 149-residue protein forms a tight dimer in solution. Mitsuba (meaning "three-leaf" in Japanese) was created by symmetry constraining the structure of a MytiLec-1 subunit, resulting in a 150-residue sequence that contains three identical tandem repeats. Mitsuba-1 was expressed and crystallised to confirm the X-ray structure matches the predicted model. Mitsuba-1 recognises cancer cells that express globotriose (Galα(1,4)Galβ(1,4)Glc) on the surface, but the cytotoxicity is abolished.
Generating and Expanding Autologous Chimeric Antigen Receptor T Cells from Patients with Acute Myeloid Leukemia
Jul 23, 2017   Methods In Molecular Biology (Clifton, N.J.)
Kenderian SS, June CH, Gill S
Generating and Expanding Autologous Chimeric Antigen Receptor T Cells from Patients with Acute Myeloid Leukemia
Jul 23, 2017
Methods In Molecular Biology (Clifton, N.J.)
Adoptive transfer of genetically engineered T cells can lead to profound and durable responses in patients with hematologic malignancies, generating enormous enthusiasm among scientists, clinicians, patients, and biotechnology companies. The success of adoptive cellular immunotherapy depends upon the ability to manufacture good quality T cells. We discuss here the methodologies and reagents that are used to generate T cells for the preclinical study of chimeric antigen receptor T cell therapy for acute myeloid leukemia (AML).
Biophysical Constraints Arising from Compositional Context in Synthetic Gene Networks
Jul 23, 2017   Cell Systems
Yeung E, Dy AJ, Martin KB, Ng AH, Del Vecchio D, Beck JL, Collins JJ, Murray RM
Biophysical Constraints Arising from Compositional Context in Synthetic Gene Networks
Jul 23, 2017
Cell Systems
Synthetic gene expression is highly sensitive to intragenic compositional context (promoter structure, spacing regions between promoter and coding sequences, and ribosome binding sites). However, much less is known about the effects of intergenic compositional context (spatial arrangement and orientation of entire genes on DNA) on expression levels in synthetic gene networks. We compare expression of induced genes arranged in convergent, divergent, or tandem orientations. Induction of convergent genes yielded up to 400% higher expression, greater ultrasensitivity, and dynamic range than divergent- or tandem-oriented genes. Orientation affects gene expression whether one or both genes are induced. We postulate that transcriptional interference in divergent and tandem genes, mediated by supercoiling, can explain differences in expression and validate this hypothesis through modeling and in vitro supercoiling relaxation experiments. Treatment with gyrase abrogated intergenic context effects, bringing expression levels within 30% of each other. We rebuilt the toggle switch with convergent genes, taking advantage of supercoiling effects to improve threshold detection and switch stability. Copyright © 2017 Elsevier Inc. All rights reserved.
The Metal Drives the Chemistry: Dual Functions of Acireductone Dioxygenase
Jul 21, 2017   Chemical Reviews
Deshpande AR, Pochapsky TC, Ringe D
The Metal Drives the Chemistry: Dual Functions of Acireductone Dioxygenase
Jul 21, 2017
Chemical Reviews
Acireductone dioxygenase (ARD) from the methionine salvage pathway (MSP) is a unique enzyme that exhibits dual chemistry determined solely by the identity of the divalent transition-metal ion (Fe2+ or Ni2+) in the active site. The Fe2+-containing isozyme catalyzes the on-pathway reaction using substrates 1,2-dihydroxy-3-keto-5-methylthiopent-1-ene (acireductone) and dioxygen to generate formate and the ketoacid precursor of methionine, 2-keto-4-methylthiobutyrate, whereas the Ni2+-containing isozyme catalyzes an off-pathway shunt with the same substrates, generating methylthiopropionate, carbon monoxide, and formate. The dual chemistry of ARD was originally discovered in the bacterium Klebsiella oxytoca, but it has recently been shown that mammalian ARD enzymes (mouse and human) are also capable of catalyzing metal-dependent dual chemistry in vitro. This is particularly interesting, since carbon monoxide, one of the products of off-pathway reaction, has been identified as an antiapoptotic molecule in mammals. In addition, several biochemical and genetic studies have indicated an inhibitory role of human ARD in cancer. This comprehensive review describes the biochemical and structural characterization of the ARD family, the proposed experimental and theoretical approaches to establishing mechanisms for the dual chemistry, insights into the mechanism based on comparison with structurally and functionally similar enzymes, and the applications of this research to the field of artificial metalloenzymes and synthetic biology.
Nephroprotective Effects of Anthocyanin from the Fruits of Panax ginseng (GFA) on Cisplatin-Induced Acute Kidney Injury in Mice
Jul 21, 2017   Phytotherapy Research : PTR
Qi ZL, Wang Z, Li W, Hou JG, Liu Y, Li XD, Li HP, Wang YP
Nephroprotective Effects of Anthocyanin from the Fruits of Panax ginseng (GFA) on Cisplatin-Induced Acute Kidney Injury in Mice
Jul 21, 2017
Phytotherapy Research : PTR
Cisplatin is an effective anticancer chemotherapeutic agent, but the use of cisplatin in the clinic is severely limited by side effects. Nephrotoxicity is a major factor that contributes to the side effects of cisplatin chemotherapy. The aim of this research was to survey the nephroprotective effects of anthocyanin from the fruits of Panax ginseng (GFA) in a murine model of cisplatin-induced acute kidney injury. We observed that pretreatment with GFA attenuated cisplatin-induced elevations in blood urea nitrogen and creatinine levels and histopathological injury induced by cisplatin. The formation of kidney malondialdehyde, heme oxygenase-1, cytochrome P450 E1 and 4-hydroxynonenal with a concomitant reduction in reduced glutathione was also inhibited by GFA, while the activities of kidney superoxide dismutase and catalase were all increased. GFA also inhibited the increase in serum tumour necrosis factor-α and interleukin-1β induced by cisplatin. In addition, the levels of induced nitric oxide synthase and cyclooxygenase-2 were suppressed by GFA. Furthermore, GFA supplementation inhibited the activation of apoptotic pathways by increasing B cell lymphoma 2 and decreasing Bcl2-associated X protein expression. In conclusion, the findings from the present investigation demonstrate that GFA pre-administration can significantly prevent cisplatin-induced nephrotoxicity, which may be related to its antioxidant, anti-apoptotic and antiinflammatory effects. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.
Expression of Recombinant Phosphoproteins for Signal Transduction Studies
Jul 21, 2017   Methods In Molecular Biology (Clifton, N.J.)
Barber KW, Rinehart J
Expression of Recombinant Phosphoproteins for Signal Transduction Studies
Jul 21, 2017
Methods In Molecular Biology (Clifton, N.J.)
Complex signaling cascades are difficult to study in vitro without phosphorylated proteins. Here, we describe a technique for the routine production of recombinant phosphoproteins by directly incorporating phosphoserine as a nonstandard amino acid. This protocol utilizes an optimized phosphoserine orthogonal translation system and an engineered strain of E. coli containing no genomic amber codons. This approach has been used to generate a variety of phosphorylated proteins to understand the role of protein phosphorylation in cell signaling.
Genome-wide landscape of position effects on heterogeneous gene expression in Saccharomyces cerevisiae
Jul 21, 2017   Biotechnology For Biofuels
Wu XL, Li BZ, Zhang WZ, Song K, Qi H, Dai JB, Yuan YJ
Genome-wide landscape of position effects on heterogeneous gene expression in Saccharomyces cerevisiae
Jul 21, 2017
Biotechnology For Biofuels
Integration of heterogeneous genes is widely applied in synthetic biology and metabolic engineering. However, knowledge about the effect of integrative position on gene expression remains limited. We established a genome-wide landscape of position effect on gene expression in Saccharomyces cerevisiae. The expression cassette of red fluorescence protein (RFP) gene was constructed and inserted at 1044 loci, which were scattered uniformly in the yeast genome. Due to the different integrative loci on the genome, the maximum relative intensity of RFP is more than 13-fold over the minimum. Plots of the number of strains to RFP relative intensity showed normal distribution, indicating significant position effect on gene expression in yeast. Furthermore, changing the promoters or reporter genes, as well as carbon sources, revealed little consequences on reporter gene expression, indicating chromosomal location is the major determinant of reporter gene expression. We have examined the position effects to integration genes expression in large number loci around whole genome in S. cerevisiae. The results could guide the design of integration loci for exogenous genes and pathways to maximize their expression in metabolic engineering.
Extensive Survey of Antibody Invariant Positions for Efficient Chemical Conjugation Using Expanded Genetic Codes
Jul 20, 2017   Bioconjugate Chemistry
Kato A, Kuratani M, Yanagisawa T, Ohtake K, Hayashi A, Amano Y, Kimura K, Yokoyama S, Sakamoto K, Shiraishi Y
Extensive Survey of Antibody Invariant Positions for Efficient Chemical Conjugation Using Expanded Genetic Codes
Jul 20, 2017
Bioconjugate Chemistry
The site-specific chemical conjugation of proteins, following synthesis with an expanded genetic code, promises to advance antibody-based technologies, including antibody drug conjugation and the creation of bispecific Fab dimers. The incorporation of non-natural amino acids into antibodies not only guarantees site specificity but also allows the use of bio-orthogonal chemistry. However, the efficiency of amino acid incorporation fluctuates significantly among different sites, thereby hampering the identification of useful conjugation sites. In this study, we applied the codon reassignment technology to achieve the robust and efficient synthesis of chemically functionalized antibodies containing Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) at defined positions. This lysine derivative has a bio-orthogonally reactive group at the end of a long side chain, enabling identification of multiple new positions in Fab-constant domains, allowing chemical conjugation with high efficiency. An X-ray crystallographic study of a Fab variant with o-Az-Z-Lys revealed high-level exposure of the azido group to solvent, with six of the identified positions subsequently used to engineer "Variabodies", a novel antibody format allowing various connections between two Fab molecules. Our findings indicated that some of the created Variabodies exhibited agonistic activity in cultured cells as opposed to the antagonistic nature of antibodies. These results showed that our approach greatly enhanced the availability of antibodies for chemical conjugation and might aid in the development of new therapeutic antibodies.
Strategies for structuring interdisciplinary education in Systems Biology: an European perspective
Jul 20, 2017   NPJ Systems Biology And Applications
Cvijovic M, Höfer T, Aćimović J, Alberghina L, Almaas E,   . . . . . .   , Stalidzans E, Stelling J, Teusink B, Workman CT, Hohmann S
Strategies for structuring interdisciplinary education in Systems Biology: an European perspective
Jul 20, 2017
NPJ Systems Biology And Applications
Systems Biology is an approach to biology and medicine that has the potential to lead to a better understanding of how biological properties emerge from the interaction of genes, proteins, molecules, cells and organisms. The approach aims at elucidating how these interactions govern biological function by employing experimental data, mathematical models and computational simulations. As Systems Biology is inherently multidisciplinary, education within this field meets numerous hurdles including departmental barriers, availability of all required expertise locally, appropriate teaching material and example curricula. As university education at the Bachelor's level is traditionally built upon disciplinary degrees, we believe that the most effective way to implement education in Systems Biology would be at the Master's level, as it offers a more flexible framework. Our team of experts and active performers of Systems Biology education suggest here (i) a definition of the skills that students should acquire within a Master's programme in Systems Biology, (ii) a possible basic educational curriculum with flexibility to adjust to different application areas and local research strengths, (iii) a description of possible career paths for students who undergo such an education, (iv) conditions that should improve the recruitment of students to such programmes and (v) mechanisms for collaboration and excellence spreading among education professionals. With the growing interest of industry in applying Systems Biology approaches in their fields, a concerted action between academia and industry is needed to build this expertise. Here we present a reflection of the European situation and expertise, where most of the challenges we discuss are universal, anticipating that our suggestions will be useful internationally. We believe that one of the overriding goals of any Systems Biology education should be a student's ability to phrase and communicate research questions in such a manner that they can be solved by the integration of experiments and modelling, as well as to communicate and collaborate productively across different experimental and theoretical disciplines in research and development.
Regulation of amino-acid metabolism controls flux to lipid accumulation in Yarrowia lipolytica
Jul 20, 2017   NPJ Systems Biology And Applications
Kerkhoven EJ, Pomraning KR, Baker SE, Nielsen J
Regulation of amino-acid metabolism controls flux to lipid accumulation in Yarrowia lipolytica
Jul 20, 2017
NPJ Systems Biology And Applications
Yarrowia lipolytica is a promising microbial cell factory for the production of lipids to be used as fuels and chemicals, but there are few studies on regulation of its metabolism. Here we performed the first integrated data analysis of Y. lipolytica grown in carbon and nitrogen limited chemostat cultures. We first reconstructed a genome-scale metabolic model and used this for integrative analysis of multilevel omics data. Metabolite profiling and lipidomics was used to quantify the cellular physiology, while regulatory changes were measured using RNAseq. Analysis of the data showed that lipid accumulation in Y. lipolytica does not involve transcriptional regulation of lipid metabolism but is associated with regulation of amino-acid biosynthesis, resulting in redirection of carbon flux during nitrogen limitation from amino acids to lipids. Lipid accumulation in Y. lipolytica at nitrogen limitation is similar to the overflow metabolism observed in many other microorganisms, e.g. ethanol production by Sacchromyces cerevisiae at nitrogen limitation.
Improvement in the production of the human recombinant enzyme N-acetylgalactosamine-6-sulfatase (rhGALNS) in Escherichia coli using synthetic biology approaches
Jul 20, 2017   Scientific Reports
Reyes LH, Cardona C, Pimentel L, Rodríguez-López A, Alméciga-Díaz CJ
Improvement in the production of the human recombinant enzyme N-acetylgalactosamine-6-sulfatase (rhGALNS) in Escherichia coli using synthetic biology approaches
Jul 20, 2017
Scientific Reports
Previously, we demonstrated production of an active recombinant human N-acetylgalactosamine-6-sulfatase (rhGALNS) enzyme in Escherichia coli as a potential therapeutic alternative for mucopolysaccharidosis IVA. However, most of the rhGALNS produced was present as protein aggregates. Here, several methods were investigated to improve production and activity of rhGALNS. These methods involved the use of physiologically-regulated promoters and alternatives to improve protein folding including global stress responses (osmotic shock), overexpression of native chaperones, and enhancement of cytoplasmic disulfide bond formation. Increase of rhGALNS activity was obtained when a promoter regulated under σ s was implemented. Additionally, improvements were observed when osmotic shock was applied. Noteworthy, overexpression of chaperones did not have any effect on rhGALNS activity, suggesting that the effect of osmotic shock was probably due to a general stress response and not to the action of an individual chaperone. Finally, it was observed that high concentrations of sucrose in conjunction with the physiological-regulated promoter proU mod significantly increased the rhGALNS production and activity. Together, these results describe advances in the current knowledge on the production of human recombinant enzymes in a prokaryotic system such as E. coli, and could have a significant impact on the development of enzyme replacement therapies for lysosomal storage diseases.
Quorum-Quenching Human Designer Cells for Closed-Loop Control of Pseudomonas aeruginosa Biofilms
Jul 13, 2017   Nano Letters
Sedlmayer F, Jaeger T, Jenal U, Fussenegger M
Quorum-Quenching Human Designer Cells for Closed-Loop Control of Pseudomonas aeruginosa Biofilms
Jul 13, 2017
Nano Letters
Current antibiotics gradually lose their efficacy against chronic Pseudomonas aeruginosa infections due to development of increased resistance mediated by biofilm formation, as well as the large arsenal of microbial virulence factors that are coordinated by the cell density-dependent phenomenon of quorum sensing. Here, we address this issue by using synthetic biology principles to rationally engineer quorum-quencher cells with closed-loop control to autonomously dampen virulence and interfere with biofilm integrity. Pathogen-derived signals dynamically activate a synthetic mammalian autoinducer sensor driving downstream expression of next-generation anti-infectives. Engineered cells were able to sensitively score autoinducer levels from P. aeruginosa clinical isolates and mount a 2-fold defense consisting of an autoinducer-inactivating enzyme to silence bacterial quorum sensing and a bipartite antibiofilm effector to dissolve the biofilm matrix. The self-guided cellular device fully cleared autoinducers, potentiated bacterial antibiotic susceptibility, substantially reduced biofilms, and alleviated cytotoxicity to lung epithelial cells. We believe this strategy of dividing otherwise coordinated pathogens and breaking up their shielded stronghold represents a blueprint for cellular anti-infectives in the postantibiotic era.
Enabling tools for high-throughput detection of metabolites: Metabolic engineering and directed evolution applications
Jul 20, 2017   Biotechnology Advances
Lin JL, Wagner JM, Alper HS
Enabling tools for high-throughput detection of metabolites: Metabolic engineering and directed evolution applications
Jul 20, 2017
Biotechnology Advances
Within the Design-Build-Test Cycle for strain engineering, rapid product detection and selection strategies remain challenging and limit overall throughput. Here we summarize a wide variety of modalities that transduce chemical concentrations into easily measured absorbance, luminescence, and fluorescence signals. Specifically, we cover protein-based biosensors (including transcription factors), nucleic acid-based biosensors, coupled enzyme reactions, bioorthogonal chemistry, and fluorescent and chromogenic dyes and substrates as modalities for detection. We focus on the use of these methods for strain engineering and enzyme discovery and conclude with remarks on the current and future state of biosensor development for application in the metabolic engineering field. Copyright © 2017 Elsevier Inc. All rights reserved.
Oligo- and dsDNA-mediated genome editing using a tetA dual selection system in Escherichia coli
Jul 18, 2017   PloS One
Ryu YS, Chandran SP, Kim K, Lee SK
Oligo- and dsDNA-mediated genome editing using a tetA dual selection system in Escherichia coli
Jul 18, 2017
PloS One
The ability to precisely and seamlessly modify a target genome is needed for metabolic engineering and synthetic biology techniques aimed at creating potent biosystems. Herein, we report on a promising method in Escherichia coli that relies on the insertion of an optimized tetA dual selection cassette followed by replacement of the same cassette with short, single-stranded DNA (oligos) or long, double-stranded DNA and the isolation of recombinant strains by negative selection using NiCl2. This method could be rapidly and successfully used for genome engineering, including deletions, insertions, replacements, and point mutations, without inactivation of the methyl-directed mismatch repair (MMR) system and plasmid cloning. The method we describe here facilitates positive genome-edited recombinants with selection efficiencies ranging from 57 to 92%. Using our method, we increased lycopene production (3.4-fold) by replacing the ribosome binding site (RBS) of the rate-limiting gene (dxs) in the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway with a strong RBS. Thus, this method could be used to achieve scarless, proficient, and targeted genome editing for engineering E. coli strains.
Molecular mechanism of environmental d-xylose perception by a XylFII-LytS complex in bacteria
Jul 18, 2017   Proceedings Of The National Academy Of Sciences Of The United States Of America
Li J, Wang C, Yang G, Sun Z, Guo H, Shao K, Gu Y, Jiang W, Zhang P
Molecular mechanism of environmental d-xylose perception by a XylFII-LytS complex in bacteria
Jul 18, 2017
Proceedings Of The National Academy Of Sciences Of The United States Of America
d-xylose, the main building block of plant biomass, is a pentose sugar that can be used by bacteria as a carbon source for bio-based fuel and chemical production through fermentation. In bacteria, the first step for d-xylose metabolism is signal perception at the membrane. We previously identified a three-component system in Firmicutes bacteria comprising a membrane-associated sensor protein (XylFII), a transmembrane histidine kinase (LytS) for periplasmic d-xylose sensing, and a cytoplasmic response regulator (YesN) that activates the transcription of the target ABC transporter xylFGH genes to promote the uptake of d-xylose. The molecular mechanism underlying signal perception and integration of these processes remains elusive, however. Here we purified the N-terminal periplasmic domain of LytS (LytSN) in a complex with XylFII and determined the conformational structures of the complex in its d-xylose-free and d-xylose-bound forms. LytSN contains a four-helix bundle, and XylFII contains two Rossmann fold-like globular domains with a xylose-binding cleft between them. In the absence of d-xylose, LytSN and XylFII formed a heterodimer. Specific binding of d-xylose to the cleft of XylFII induced a large conformational change that closed the cleft and brought the globular domains closer together. This conformational change led to the formation of an active XylFII-LytSN heterotetramer. Mutations at the d-xylose binding site and the heterotetramer interface diminished heterotetramer formation and impaired the d-xylose-sensing function of XylFII-LytS. Based on these data, we propose a working model of XylFII-LytS that provides a molecular basis for d-xylose utilization and metabolic modification in bacteria.
Ethylene induces combinatorial effects of histone H3 acetylation in gene expression in Arabidopsis
Jul 18, 2017   BMC Genomics
Wang L, Zhang F, Rode S, Chin KK, Ko EE, Kim J, Iyer VR, Qiao H
Ethylene induces combinatorial effects of histone H3 acetylation in gene expression in Arabidopsis
Jul 18, 2017
BMC Genomics
Histone acetylation and deacetylation are essential for gene regulation and have been implicated in the regulation of plant hormone responses. Many studies have indicated the role of histone acetylation in ethylene signaling; however, few studies have investigated how ethylene signaling regulates the genomic landscape of chromatin states. Recently, we found that ethylene can specifically elevate histone H3K14 acetylation and the non-canonical histone H3K23 acetylation in etiolated seedlings and the gene activation is positively associated with the elevation of H3K14Ac and H3K23Ac in response to ethylene. To assess the role of H3K9, H3K14, and H3K23 histone modifications in the ethylene response, we examined how ethylene regulates histone acetylation and the transcriptome at global level and in ethylene regulated genes both in wild type (Col-0) and ein2-5 seedlings. Our results revealed that H3K9Ac, H3K14Ac, and H3K23Ac are preferentially enriched around the transcription start sites and are positively correlated with gene expression levels in Col-0 and ein2-5 seedlings both with and without ethylene treatment. In the absence of ethylene, no combinatorial effect of H3K9Ac, H3K14Ac, and H3K23Ac on gene expression was detected. In the presence of ethylene, however, combined enrichment of the three histone acetylation marks was associated with high gene expression levels, and this ethylene-induced change was EIN2 dependent. In addition, we found that ethylene-regulated genes are expressed at medium or high levels, and a group of ethylene regulated genes are marked by either one of H3K9Ac, H3K14Ac or H3K23Ac. In this group of genes, the levels of H3K9Ac were altered by ethylene, but in the absence of ethylene the levels of H3K9Ac and peak breadths are distinguished in up- and down- regulated genes. In the presence of ethylene, the changes in the peak breadths and levels of H3K14Ac and H3K23Ac are required for the alteration of gene expressions. Our study reveals that the plant hormone ethylene induces combinatorial effects of H3K9Ac, K14Ac and K23Ac histone acetylation in gene expression genome widely. Further, for a group of ethylene regulated genes, in the absence of ethylene the levels and the covered breadths of H3K9Ac are the preexist markers for distinguishing up- and down- regulated genes, the change in the peak breadths and levels of H3K14Ac and H3K23Ac are required for the alteration of gene expression in the presence of ethylene.
Engineering Strategies to Decode and Enhance the Genomes of Coral Symbionts
Jul 17, 2017   Frontiers In Microbiology
Levin RA, Voolstra CR, Agrawal S, Steinberg PD, Suggett DJ, van Oppen MJH
Engineering Strategies to Decode and Enhance the Genomes of Coral Symbionts
Jul 17, 2017
Frontiers In Microbiology
Elevated sea surface temperatures from a severe and prolonged El Niño event (2014-2016) fueled by climate change have resulted in mass coral bleaching (loss of dinoflagellate photosymbionts, Symbiodinium spp., from coral tissues) and subsequent coral mortality, devastating reefs worldwide. Genetic variation within and between Symbiodinium species strongly influences the bleaching tolerance of corals, thus recent papers have called for genetic engineering of Symbiodinium to elucidate the genetic basis of bleaching-relevant Symbiodinium traits. However, while Symbiodinium has been intensively studied for over 50 years, genetic transformation of Symbiodinium has seen little success likely due to the large evolutionary divergence between Symbiodinium and other model eukaryotes rendering standard transformation systems incompatible. Here, we integrate the growing wealth of Symbiodinium next-generation sequencing data to design tailored genetic engineering strategies. Specifically, we develop a testable expression construct model that incorporates endogenous Symbiodinium promoters, terminators, and genes of interest, as well as an internal ribosomal entry site from a Symbiodinium virus. Furthermore, we assess the potential for CRISPR/Cas9 genome editing through new analyses of the three currently available Symbiodinium genomes. Finally, we discuss how genetic engineering could be applied to enhance the stress tolerance of Symbiodinium, and in turn, coral reefs.
Ciprofloxacin-nitroxide hybrids with potential for biofilm control
Jul 14, 2017   European Journal Of Medicinal Chemistry
Verderosa AD, de la Fuente-Núñez C, Mansour SC, Cao J, Lu TK, Hancock REW, Fairfull-Smith KE
Ciprofloxacin-nitroxide hybrids with potential for biofilm control
Jul 14, 2017
European Journal Of Medicinal Chemistry
As bacterial biofilms display extreme tolerance to conventional antibiotic treatments, it has become imperative to develop new antibacterial strategies with alternative mechanisms of action. Herein, we report the synthesis of a series of ciprofloxacin-nitroxide conjugates and their corresponding methoxyamine derivatives in high yield. This was achieved by linking various nitroxides or methoxyamines to the secondary amine of the piperazine ring of ciprofloxacin using amide bond coupling. Biological evaluation of the prepared compounds on preformed P. aeruginosa biofilms in flow cells revealed substantial dispersal with ciprofloxacin-nitroxide hybrid 25, and virtually complete killing and removal (94%) of established biofilms in the presence of ciprofloxacin-nitroxide hybrid 27. Compounds 25-28 were shown to be non-toxic in both human embryonic kidney 293 (HEK 293) cells and human muscle rhabdomyosarcoma (RD) cells at concentrations up to 40 μM. Significantly, these hybrids demonstrate the potential of antimicrobial-nitroxide agents to overcome the resistance of biofilms to antimicrobials via stimulation of biofilm dispersal or through direct cell killing. Crown Copyright © 2017. Published by Elsevier Masson SAS. All rights reserved.
Advances in design of protein folds and assemblies
Jul 14, 2017   Current Opinion In Chemical Biology
Ljubetič A, Gradišar H, Jerala R
Advances in design of protein folds and assemblies
Jul 14, 2017
Current Opinion In Chemical Biology
Conceptual and computational advances triggered an explosion of designed protein structures in the recent years. Various protein fold geometries have been robustly designed with atomic accuracy, including protein folds unseen in nature. The same principles and tools have been extended to design multi-chain assemblies. By exploiting symmetry, mega-Dalton structures have been created with exciting potential applications for synthetic biology. In this review we focus on design of single chain and multi polypeptide chain assemblies of defined size and composition. Several innovative strategies have been developed to create de novo protein assemblies, with the two main approaches to the design of multi-chain assemblies being genetic fusion of interacting modules and engineering of novel protein-protein interfaces. Copyright © 2017. Published by Elsevier Ltd.
biochem4j: Integrated and extensible biochemical knowledge through graph databases
Jul 14, 2017   PloS One
Swainston N, Batista-Navarro R, Carbonell P, Dobson PD, Dunstan M, Jervis AJ, Vinaixa M, Williams AR, Ananiadou S, Faulon JL, Mendes P, Kell DB, Scrutton NS, Breitling R
biochem4j: Integrated and extensible biochemical knowledge through graph databases
Jul 14, 2017
PloS One
Biologists and biochemists have at their disposal a number of excellent, publicly available data resources such as UniProt, KEGG, and NCBI Taxonomy, which catalogue biological entities. Despite the usefulness of these resources, they remain fundamentally unconnected. While links may appear between entries across these databases, users are typically only able to follow such links by manual browsing or through specialised workflows. Although many of the resources provide web-service interfaces for computational access, performing federated queries across databases remains a non-trivial but essential activity in interdisciplinary systems and synthetic biology programmes. What is needed are integrated repositories to catalogue both biological entities and-crucially-the relationships between them. Such a resource should be extensible, such that newly discovered relationships-for example, those between novel, synthetic enzymes and non-natural products-can be added over time. With the introduction of graph databases, the barrier to the rapid generation, extension and querying of such a resource has been lowered considerably. With a particular focus on metabolic engineering as an illustrative application domain, biochem4j, freely available at http://biochem4j.org, is introduced to provide an integrated, queryable database that warehouses chemical, reaction, enzyme and taxonomic data from a range of reliable resources. The biochem4j framework establishes a starting point for the flexible integration and exploitation of an ever-wider range of biological data sources, from public databases to laboratory-specific experimental datasets, for the benefit of systems biologists, biosystems engineers and the wider community of molecular biologists and biological chemists.
More than just a gut feeling: constraint-based genome-scale metabolic models for predicting functions of human intestinal microbes
Jul 14, 2017   Microbiome
van der Ark KCH, van Heck RGA, Martins Dos Santos VAP, Belzer C, de Vos WM
More than just a gut feeling: constraint-based genome-scale metabolic models for predicting functions of human intestinal microbes
Jul 14, 2017
Microbiome
The human gut is colonized with a myriad of microbes, with substantial interpersonal variation. This complex ecosystem is an integral part of the gastrointestinal tract and plays a major role in the maintenance of homeostasis. Its dysfunction has been correlated to a wide array of diseases, but the understanding of causal mechanisms is hampered by the limited amount of cultured microbes, poor understanding of phenotypes, and the limited knowledge about interspecies interactions. Genome-scale metabolic models (GEMs) have been used in many different fields, ranging from metabolic engineering to the prediction of interspecies interactions. We provide showcase examples for the application of GEMs for gut microbes and focus on (i) the prediction of minimal, synthetic, or defined media; (ii) the prediction of possible functions and phenotypes; and (iii) the prediction of interspecies interactions. All three applications are key in understanding the role of individual species in the gut ecosystem as well as the role of the microbiota as a whole. Using GEMs in the described fashions has led to designs of minimal growth media, an increased understanding of microbial phenotypes and their influence on the host immune system, and dietary interventions to improve human health. Ultimately, an increased understanding of the gut ecosystem will enable targeted interventions in gut microbial composition to restore homeostasis and appropriate host-microbe crosstalk.
Molecular tools and emerging strategies for deep genetic/genomic refactoring of Pseudomonas
Jul 24, 2017   Current Opinion In Biotechnology
Martínez-García E, de Lorenzo V
Molecular tools and emerging strategies for deep genetic/genomic refactoring of Pseudomonas
Jul 24, 2017
Current Opinion In Biotechnology
The interest of the genus Pseudomonas largely relies on the virulence of some of its species for plants and animals (including humans). Yet, pathogenic features of some isolates coexist with others often present in environmental variants that promote plant growth and degrade chemical pollutants. Many of these traits can be traced to the intrinsic properties of the genomic chassis of this genus along with distinct genetic parts and devices. With the tools of Synthetic Biology these can be enhanced and/or repurposed for the sake of biological control, environmental remediation and whole-cell biocatalysis. In this article we take stock of both conceptual and technological developments that have allowed the virtual domestication of Pseudomonas (in particular P. putida) as a major biotechnological workhorse with a range of applications of industrial interest. Adoption of a suite of compositional and measurement standards is advocated for bringing Pseudomonas-based genetic engineering to a superior level of development. Copyright © 2017. Published by Elsevier Ltd.
Unique Thermal Stability of Unnatural Hydrophobic Ds Bases in Double-Stranded DNAs
Jul 13, 2017   ACS Synthetic Biology
Kimoto M, Hirao I
Unique Thermal Stability of Unnatural Hydrophobic Ds Bases in Double-Stranded DNAs
Jul 13, 2017
ACS Synthetic Biology
Genetic alphabet expansion technology, the introduction of unnatural bases or base pairs into replicable DNA, has rapidly advanced as a new synthetic biology area. A hydrophobic unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) exhibited high fidelity as a third base pair in PCR. SELEX methods using the Ds-Px pair enabled high-affinity DNA aptamer generation, and introducing a few Ds bases into DNA aptamers extremely augmented their affinities and selectivities to target proteins. Here, to further scrutinize the functions of this highly hydrophobic Ds base, the thermal stabilities of double-stranded DNAs (dsDNA) containing a noncognate Ds-Ds or G-Ds pair were examined. The thermal stability of the Ds-Ds self-pair was as high as that of the natural G-C pair, and apart from the generally higher stability of the G-C pair than that of the A-T pair, most of the 5'-pyrimidine-Ds-purine-3' sequences, such as CDsA and TDsA, exhibited higher stability than the 5'-purine-Ds-pyrimidine-3' sequences, such as GDsC and ADsC, in dsDNAs. This trait enabled the GC-content-independent control of the thermal stability of the designed dsDNA fragments. The melting temperatures of dsDNA fragments containing the Ds-Ds pair can be predicted from the nearest-neighbor parameters including the Ds base. In addition, the noncognate G-Ds pair can efficiently distinguish its neighboring cognate natural base pairs from noncognate pairs. We demonstrated that real-time PCR using primers containing Ds accurately detected a single-nucleotide mismatch in target DNAs. These unique properties of the Ds base that affect the stabilities of the neighboring base pairs could impart new functions to DNA molecules and technologies.
A Synthetic Circuit for Mercury Bioremediation Using Self-Assembling Functional Amyloids
Jul 24, 2017   ACS Synthetic Biology
Tay PKR, Nguyen PQ, Joshi NS
A Synthetic Circuit for Mercury Bioremediation Using Self-Assembling Functional Amyloids
Jul 24, 2017
ACS Synthetic Biology
Synthetic biology approaches to bioremediation are a key sustainable strategy to leverage the self-replicating and programmable aspects of biology for environmental stewardship. The increasing spread of anthropogenic mercury pollution into our habitats and food chains is a pressing concern. Here, we explore the use of programmed bacterial biofilms to aid in the sequestration of mercury. We demonstrate that by integrating a mercury-responsive promoter and an operon encoding a mercury-absorbing self-assembling extracellular protein nanofiber, we can engineer bacteria that can detect and sequester toxic Hg2+ ions from the environment. This work paves the way for the development of on-demand biofilm living materials that can operate autonomously as heavy-metal absorbents.

The link you entered does not seem to be valid

Please make sure the link points to nature.com contains a valid shared_access_token

Downloading PDF to your library...

Uploading PDF...

PDF uploading

Delete tag: