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  • Biotechnology & Synthetic Biology  (41)
  • Oxford University Press  (41)
  • 2015-2019  (41)
  • 1945-1949
  • 1
    Publication Date: 2016-05-12
    Description: In this study, we isolated 15 endophytic fungi from five Sudanese medicinal plants. Each fungal endophytic strain was identified by sequencing of internal transcribed spacer (ITS) regions of rDNA. Ethyl acetate extracts were prepared from each endophyte cultivated in vitro and tested for their respective antibacterial activities and antiproliferative activities against human cancer cells. Antibacterial screening was carried out against two bacterial strains: Gram-negative Escherichia coli and Gram-positive methicillin-resistant Staphylococcus aureus , by the broth dilution method. Cell viability was evaluated by the MTT procedure after exposure of MCF7 breast cancer cells and HT29 or HCT116 human colon adenocarcinoma cells to each endophytic extract. Of interest, Byssochlamys spectabilis isolated from Euphorbia prostata showed cytotoxicity (IC 50 = 1.51 ± 0.2 μg mL –1 ) against MCF7 cells, but had a low effect against HT29 or HCT116 cells (IC 50 〉 20 μg mL –1 ). Cladosporium cladosporioides 2, isolated from Vernonia amygdalina leaves, showed antiproliferative activities against MCF7 cells (IC 50 = 10.5 ± 1.5 μg mL –1 ) only. On the other hand, B. spectabilis and Alternaria sp. extract had antibacterial activities against the S. aureus strain. The findings of this work revealed that endophytic fungi associated with medicinal plants from Sudan could be considered as an attractive source of new therapeutic compounds.
    Keywords: Biotechnology & Synthetic Biology
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  • 2
    Publication Date: 2016-04-08
    Description: Resveratrol is a well-known triphenolic natural product present in red wine. For its contribution to human health, the demand for resveratrol as a food and nutrition supplement has increased significantly. In recent years, the rapid development of synthetic biology has promoted extensive work to increase the production of resveratrol in microbes. However, supplementation of expensive phenylpropanoic precursors was required in current engineered strains. Here, we first utilized the site-specific integration strategy to produce resveratrol in Escherichia coli . The genes tal , 4cl and sts were site-specific integrated into the loci of genes tyrR and trpED in the chromosome of E. coli BW25113 (DE3). The final strain was capable of producing 4.612 mg L –1 of resveratrol from glucose.
    Keywords: Biotechnology & Synthetic Biology
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  • 3
    Publication Date: 2016-04-08
    Description: While extensively studied in several model organisms, the role of small, non-coding RNAs in the stress response remains largely unexplored in Clostridium organisms. About 100 years after the first industrial Acetone–Butanol–Ethanol fermentation process, based on the Weizmann Clostridium acetobutylicum strain, strain tolerance to butanol remains a crucial factor limiting the economics of the process. Several studies have examined the response of this organism to metabolite stress, and several genes have been engaged to impart enhanced tolerance, but no sRNAs have yet been directly engaged in this task. We show that the two stress-responsive sRNAs, 6S and tmRNA, upon overexpression impart tolerance to butanol as assessed by viability assays under process-relevant conditions. 6S overexpression enhances cell densities as well as butanol titres. We discuss the likely mechanisms that these two sRNAs might engage in this tolerance phenotype. Our data support the continued exploration of sRNAs as a basis for engineering enhanced tolerance and enhanced solvent production, especially because sRNA-based strategies impose a minimal metabolic burden on the cells.
    Keywords: Biotechnology & Synthetic Biology
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  • 4
    Publication Date: 2016-07-02
    Description: Ten indole alkaloids were obtained from the marine sponge-associated fungus Neosartorya siamensis KUFA 0017. We studied the antimicrobial properties of these and of three other compounds previously isolated from the soil fungus N. siamensis KUFC 6349. Only neofiscalin A showed antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE); with a minimum inhibitory concentration (MIC) of 8 μg mL –1 against both strains. Another compound, fiscalin C, presented synergistic activity against MRSA when combined with oxacillin, although alone showed no antibacterial effect. Moreover, neofiscalin A, when present at sub-MICs, hampered the ability of both MRSA and VRE strains to form a biofilm. Additionally, the biofilm inhibitory concentration values of neofiscalin A against the MRSA and VRE isolates were 96 and 80 μg mL –1 , respectively. At a concentration of 200 μg mL –1 , neofiscalin A was able to reduce the metabolic activity of the biofilms by ~50%. One important fact is that our results also showed that neofiscalin A had no cytotoxicity against a human brain capillary endothelial cell line.
    Keywords: Biotechnology & Synthetic Biology
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  • 5
    Publication Date: 2016-07-02
    Description: During unconventional protein secretion (UPS), proteins do not pass through the classical endoplasmic reticulum (ER)–Golgi-dependent pathway, but are transported to the cell membrane via alternative routes. One type of UPS is dependent on several autophagy-related (Atg) proteins in yeast and mammalian cells, but mechanisms for unconventional secretion are largely unknown for filamentous fungi. In this study, we investigated whether the autophagy machinery is used for UPS in the filamentous fungus Aspergillus niger . An aspartic protease, which we called PepN, was identified as being likely to be secreted unconventionally, as this protein is highly abundant in culture filtrates during carbon starvation while it lacks a conventional N-terminal secretion sequence. We analysed the presence of PepN in the culture filtrates of carbon starved wild-type, atg1 and atg8 deletion mutant strains by Western blot analysis and by secretome analysis using nanoLC-ESI-MS/MS (wild-type and atg8 deletion mutant). Besides the presence of carbohydrate-active enzymes and other types of proteases, PepN was abundantly found in culture filtrates of both wild-type and atg deletion strains, indicating that the secretion of PepN is independent of the autophagy machinery in A. niger and hence most likely occurs via a different mechanism.
    Keywords: Biotechnology & Synthetic Biology
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  • 6
    Publication Date: 2016-08-27
    Description: Actin-like MreB paralogs play important roles in cell shape maintenance, cell wall synthesis and the regulation of the D,L-endopeptidases, CwlO and LytE. The gram-positive bacteria, Bacillus amyloliquefaciens LL3, is a poly--glutamic acid (-PGA) producing strain that contains three MreB paralogs: MreB, Mbl and MreBH. In B. amyloliquefaciens , CwlO and LytE can degrade -PGA. In this study, we aimed to test the hypothesis that modulating transcript levels of MreB paralogs would alter the synthesis and degradation of -PGA. The results showed that overexpression or inhibition of MreB, Mbl or MreBH had distinct effects on cell morphology and the molecular weight of the -PGA products. In fermentation medium, cells of mreB inhibition mutant were 50.2% longer than LL3, and the -PGA titer increased by 55.7%. However, changing the expression level of mbl showed only slight effects on the morphology, -PGA molecular weight and titer. In the mreBH inhibition mutant, -PGA production and its molecular weight increased by 56.7% and 19.4%, respectively. These results confirmed our hypothesis that suppressing the expression of MreB paralogs might reduce -PGA degradation, and that improving the cell size could strengthen -PGA synthesis. This is the first report of enhanced -PGA production via suppression of actin-like MreB paralogs.
    Keywords: Biotechnology & Synthetic Biology
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  • 7
    Publication Date: 2016-06-08
    Description: Microbial production of acetone and butanol was one of the first large-scale industrial fermentation processes of global importance. During the first part of the 20th century, it was indeed the second largest fermentation process, superseded in importance only by the ethanol fermentation. After a rapid decline after the 1950s, acetone-butanol-ethanol (ABE) fermentation has recently gained renewed interest in the context of biorefinery approaches for the production of fuels and chemicals from renewable resources. The availability of new methods and knowledge opens many new doors for industrial microbiology, and a comprehensive view on this process is worthwhile due to the new interest. This thematic issue of FEMS Microbiology Letters, dedicated to the 100th anniversary of the first industrial exploitation of Chaim Weizmann's ABE fermentation process, covers the main aspects of old and new developments, thereby outlining a model development in biotechnology. All major aspects of industrial microbiology are exemplified by this single process. This includes new technologies, such as the latest developments in metabolic engineering, the exploitation of biodiversity and discoveries of new regulatory systems such as for microbial stress tolerance, as well as technological aspects, such as bio- and down-stream processing.
    Keywords: Biotechnology & Synthetic Biology
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  • 8
    Publication Date: 2016-06-02
    Description: Food-grade lactic acid bacteria, such as lactobacilli, represent good candidates for the development of mucosal vectors. Indeed, they are generally recognized as safe microorganisms and some strains display beneficial effects (probiotics). In this study, we described a new lactobacilli in vivo expression (LIVE) system for the production and delivery of therapeutic molecules at mucosal surfaces. The versatility and functionality of this system was successfully validated in several lactobacilli species; furthermore, we assessed in vivo LIVE system in two different mouse models of human pathologies: (i) a model of therapy against intestinal inflammation (inflammatory bowel diseases) and (ii) a model of vaccination against dental caries. We demonstrated that Lactobacillus gasseri expressing the anti-inflammatory cytokine IL-10 under LIVE system efficiently delivered the recombinant protein at mucosal surfaces and display anti-inflammatory effects. In the vaccination model against caries, LIVE system allowed the heterologous expression of Streptococcus mutans antigen GbpB by L. gasseri , leading to a stimulation of the host immune response.
    Keywords: Biotechnology & Synthetic Biology
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  • 9
    Publication Date: 2016-06-02
    Description: The microbial fermentation process is one of the sustainable and environment-friendly ways to produce 1-butanol and other bio-based chemicals. The success of the fermentation process greatly relies on the choice of bioreactors and the separation methods. In this review, the history and the performance of bioreactors for the acetone–butanol–ethanol (ABE) fermentation is discussed. The subject is then focused on in situ product recovery (ISPR) techniques, particularly for the integrated extraction-gas stripping. The usefulness of this promising hybrid ISPR device is acknowledged by its incorporation with batch, fed-batch and continuous processes to improve the performance of ABE fermentation.
    Keywords: Biotechnology & Synthetic Biology
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  • 10
    Publication Date: 2016-01-29
    Description: Anaerobic Clostridium spp. is an important bioproduction microbial genus that can produce solvents and utilize a broad spectrum of substrates including cellulose and syngas. Genome-scale metabolic (GSM) models are increasingly being put forth for various clostridial strains to explore their respective metabolic capabilities and suitability for various bioconversions. In this study, we have selected representative GSM models for six different clostridia ( Clostridium acetobutylicum , C. beijerinckii , C. butyricum , C. cellulolyticum , C. ljungdahlii and C. thermocellum ) and performed a detailed model comparison contrasting their metabolic repertoire. We also discuss various applications of these GSM models to guide metabolic engineering interventions as well as assessing cellular physiology.
    Keywords: Biotechnology & Synthetic Biology
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  • 11
    Publication Date: 2016-03-02
    Description: Biobutanol is a next-generation liquid biofuel with properties akin to those of gasoline. There is a widespread effort to commercialize biobutanol production from agricultural residues, such as corn stover, which do not compete with human and animal foods. This pursuit is backed by extensive government mandates to expand alternative energy sources. This review provides an overview of research on biobutanol production using corn stover feedstock. Structural composition, pretreatment, sugar yield (following pretreatment and hydrolysis) and generation of lignocellulose-derived microbial inhibitory compounds (LDMICs) from corn stover are discussed. The review also discusses different Clostridium species and strains employed for biobutanol production from corn stover-derived sugars with respect to solvent yields, tolerance to LDMICs and in situ solvent recovery (integrated fermentation). Further, the economics of cellulosic biobutanol production are highlighted and compared to corn starch-derived ethanol and gasoline. As discussed herein, the economic competitiveness of biobutanol production from corn stover largely depends on feedstock processing and fermentation process design.
    Keywords: Biotechnology & Synthetic Biology
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  • 12
    Publication Date: 2016-02-20
    Description: Biological production in heterologous hosts is of interest for the production of the C4 alcohol (butanol) and other chemicals. However, some hurdles need to be overcome in order to achieve an economically viable process; these include avoiding the consumption of butanol and maintaining tolerance to this solvent during production. Pseudomonas putida is a potential host for solvent production; in order to further adapt P. putida to this role, we generated mini-Tn 5 mutant libraries in strain BIRD-1 that do not consume butanol. We analyzed the insertion site of the mini-Tn 5 in a mutant that was deficient in assimilation of butanol using arbitrary PCR followed by Sanger sequencing and found that the transposon was inserted in the malate synthase B gene. Here, we show that in a second round of mutagenesis a double mutant unable to take up butanol had an insertion in a gene coding for a multisensor hybrid histidine kinase. The genetic context of the histidine kinase sensor revealed the presence of a set of genes potentially involved in butanol assimilation; qRT-PCR analysis showed induction of this set of genes in the wild type and the malate synthase mutant but not in the double mutant.
    Keywords: Biotechnology & Synthetic Biology
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  • 13
    Publication Date: 2016-02-20
    Description: The heavy dependence on petroleum-derived fuel has raised concerns about energy sustainability and climate change, which have prompted researchers to explore fuel production from renewable sources. 1-Butanol and isobutanol are promising biofuels that have favorable properties and can also serve as solvents or chemical feedstocks. Microbial production of these alcohols provides great opportunities to access a wide spectrum of renewable resources. In recent years, research has improved the native 1-butanol production and has engineered isobutanol production in various organisms to explore metabolic diversity and a broad range of substrates. This review focuses on progress in metabolic engineering for the production of these two compounds using various resources.
    Keywords: Biotechnology & Synthetic Biology
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  • 14
    Publication Date: 2016-02-20
    Description: Biobased production of butanol promises a more sustainable route for industrial production. However, butanol toxicity remains a barrier for achieving high product titers. Investigation into butanol stress has shed some light on its modes of toxicity. Unfortunately, there still remain significant shortfalls in our understanding of the complex interactions of butanol with cells. To address this knowledge gap, a diverse range of tools have been employed to gain a better understanding of the adverse effects of butanol on the cell. These findings have lead to the identification of possible molecular mechanisms associated with butanol tolerance, which can be harnessed for future strain development efforts. This review focuses on recent efforts to address the toxicity of butanol in microbial producers and offers some perspectives on the future direction of this research sector.
    Keywords: Biotechnology & Synthetic Biology
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  • 15
    Publication Date: 2016-02-25
    Description: Biobutanol outperforms bioethanol as an advanced biofuel, but is not economically competitive in terms of its titer, yield and productivity associated with feedstocks and energy cost. In this work, the synergistic effect of calcium and zinc was investigated in the acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum using glucose, xylose and glucose/xylose mixtures as carbon source(s). Significant improvements associated with enhanced glucose/xylose utilization, cell growth, acids re-assimilation and butanol biosynthesis were achieved. Especially, the maximum butanol and ABE production of 16.1 and 25.9 g L –1 were achieved from 69.3 g L –1 glucose with butanol/ABE productivities of 0.40 and 0.65 g L –1 h –1 compared to those of 11.7 and 19.4 g/L with 0.18 and 0.30 g L –1 h –1 obtained in the control respectively without any supplement. More importantly, zinc was significantly involved in the butanol tolerance based on the improved xylose utilization under various butanol-shock conditions (2, 4, 6, 8 and 10 g L –1 butanol). Under the same conditions, calcium and zinc co-supplementation led to the best xylose utilization and butanol production. These results suggested that calcium and zinc could play synergistic roles improving ABE fermentation by C. acetobutylicum .
    Keywords: Biotechnology & Synthetic Biology
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  • 16
    Publication Date: 2016-02-07
    Description: Pseudomonas putida KT2440 is a saprophytic, environmental microorganism that plays important roles in the biodegradation of environmental toxic compounds and production of polymers, chemicals and secondary metabolites. Gene deletion of KT2440 usually involves cloning of the flanking homologous fragments of the gene of interest into a suicide vector followed by transferring into KT2440 via triparental conjugation. Selection and counterselection steps are then employed to generate gene deletion mutant. However, these methods are tedious and are not suitable for the manipulation of multiple genes simultaneously. Herein, a two-step, markerless gene deletion method is presented. First, homologous armsflanked loxP-neo-loxP was knocked-in to replace the gene of interest, then the kanamycin resistance marker is removed by Cre recombinase catalyzed site-specific recombination. Both two-plasmid and one-plasmid gene systems were established. MekR/P mekA regulated gene expression system was found to be suitable for tight Cre expression in one-plasmid deletion system. The straightforward, time saving and highly efficient markerless gene deletion strategy has the potential to facilitate the genetics and functional genomics study of P. putida KT2440.
    Keywords: Biotechnology & Synthetic Biology
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  • 17
    Publication Date: 2016-03-02
    Description: Flow cytometry, in combination with fluorescent staining, was used to evaluate population heterogeneity in acetone-butanol–ethanol fermentation that was carried out with type strain Clostridium beijerinckii NCIMB 8052 and non-type C. pasteurianum NRRL B-598. A combination of propidium iodide (PI) and carboxyfluorescein diacetate (CFDA), PI plus Syto-9 and bis-oxonol (BOX) alone were employed to distinguish between active and damaged cells together with simultaneous detection of spores. These strategies provided valuable information on the physiological state of clostridia. CFDA and PI staining gave the best separation of four distinct subpopulations of enzymatically active cells, doubly stained cells, damaged cells and spores. Proportional representation of cells in particular sub-regions correlated with growth characteristics, fermentation parameters such as substrate consumption and product formation in both species under different cultivation conditions.
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  • 18
    Publication Date: 2016-02-03
    Description: Simultaneous saccharification and fermentation (SSF) process was applied for biobutanol production by Clostridium saccharobutylicum DSM 13864 from corn stover (CS). The key influential factors in SSF process, including corn steep liquor concentration, dry biomass and enzyme loading, SSF temperature, inoculation size and pre-hydrolysis time were optimized. In 5-L bioreactor with SSF process, butanol titer and productivity of 12.3 g/L and 0.257 g/L/h were achieved at 48 h, which were 20.6% and 21.2% higher than those in separate hydrolysis and fermentation (SHF), respectively. The butanol yield reached 0.175 g/g pretreated CS in SSF, representing 50.9% increase than that in SHF (0.116 g/g pretreated CS). This study proves the feasibility of efficient and economic production of biobutanol from CS by SSF.
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  • 19
    Publication Date: 2016-03-13
    Description: We previously reported that the β-1,4-Mannanase ( manB ) gene from Bacillus pumilus functions as a good reporter gene in Lactobacillus casei . Two vectors were constructed. One carries the signal peptide of secretion protein Usp45 (SP Usp45 ) from Lactococcus lactis (pELSH), and the other carries the full-length S-layer protein, SlpA, from L. acidophilus (pELWH). In this work, another vector, pELSPH, was constructed to include the signal peptide of protein SlpA (SP SlpA ), and the capacity of all three vectors to drive expression of the manB gene in L. casei was evaluated. The results showed that SP Usp45 is functionally recognized and processed by the L. casei secretion machinery. The SP Usp45 -mediated secretion efficiency was ~87%, and SP SlpA drove the export of secreted ManB with ~80% efficiency. SP SlpA secretion was highly efficient, and expressed SlpA was anchored to the cell wall by an unknown secretion mechanism. Full-length SlpA drove the cell wall-anchored expression of an SlpA-ManB fusion protein but at a much lower level than that of protein SlpA.
    Keywords: Biotechnology & Synthetic Biology
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  • 20
    Publication Date: 2016-03-13
    Description: The porin MspA of Mycobacterium smegmatis is a biological nanopore used for DNA sequencing. The octameric MspA pore can be isolated from M. smegmatis in milligram quantities, is extremely stable against denaturation and rapidly inserts into lipid membranes. Here, we show that MspA pores composed of different Msp subunits are formed in M. smegmatis and that hetero-oligomers of different Msp monomers increase the heterogeneity of MspA pores designed for DNA sequencing. To improve the quality of preparations of mutant MspA proteins, all four msp genes were deleted from the M. smegmatis genome after insertion of an inducible porin gene from M. tuberculosis. In the msp quadruple mutant M. smegmatis ML712 no Msp porins were detected and mutant MspA proteins were produced at wild-type levels. Lipid bilayer experiments demonstrated that MspA pores isolated from ML712 formed functional channels and had a narrower conductance distribution than pores purified from M. smegmatis with background msp expression. Thus, the M. smegmatis msp quadruple mutant improves the homogeneity of MspA pores designed for DNA sequencing and might also facilitate the identification and functional characterization of other mycobacterial pore proteins.
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  • 21
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    Oxford University Press
    Publication Date: 2016-03-04
    Description: Mostly, butanol is formed as a product by saccharolytic anaerobes, employing the so-called ABE fermentation (for acetone–butanol–ethanol). However, this alcohol can also be produced from gaseous substrates such as syn(thesis) gas (major components are carbon monoxide and hydrogen) by autotrophic acetogens. In view of economic considerations, a biotechnological process based on cheap and abundant gases such as CO and CO 2 as a carbon source is preferable to more expensive sugar or starch fermentation. In addition, any conflict for use of substrates that can also serve as human nutrition is avoided. Natural formation of butanol has been found with, e.g. Clostridium carboxidivorans , while metabolic engineering for butanol production was successful using, e.g. C. ljungdahlii . Production of butanol from CO 2 under photoautotrophic conditions was also possible by recombinant DNA construction of a respective cyanobacterial Synechococcus sp. PCC 7942 strain.
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  • 22
    Publication Date: 2016-05-20
    Description: Lichen-forming fungi and extracts derived from them have been used as alternative medicine sources for millennia and recently there has been a renewed interest in their known bioactive properties for anticancer agents, cosmetics and antibiotics. Although lichen-forming fungus-derived compounds are biologically and commercially valuable, few studies have been performed to determine their modes of action. This study used chemical-genetic and chemogenomic high-throughput analyses to gain insight into the modes of action of Caloplaca flavoruscens extracts. High-throughput screening of 575 lichen extracts was performed and 39 extracts were identified which inhibited yeast growth. A C. flavoruscens extract was selected as a promising antifungal and was subjected to genome-wide haploinsufficiency profiling and homozygous profiling assays. These screens revealed that yeast deletion strains lacking Rsc8, Pro1 and Toa2 were sensitive to three concentrations (IC 25.5 , IC 25 and IC 50 , respectively) of C. flavoruscens extract. Gene-enrichment analysis of the data showed that C. flavoruscens extracts appear to perturb transcription and chromatin remodeling.
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  • 23
    Publication Date: 2016-05-20
    Description: Plant lectins, which are proteins/glycoproteins present in a wide range of vegetables, fruits, cereals and beans, are resistant to digestive enzymes and food cooking temperatures. They bind reversibly to specific glycosidic residues expressed on the membrane of intestinal epithelial cells (IEC) and cause anti-nutritional effects in humans and animals. Soybean lectin (SBA) has been detected in poultry diets, and its ability to bind to the intestinal epithelium has been reported. The development of new methods for removing SBA from feeds or to prevent interaction with the intestinal mucosa is of interest. In this study, the in vitro cytotoxicity of SBA on IEC of chicks was demonstrated for the first time. The LD 50 , assessed after 2 h exposure of IEC to SBA, was 6.13 μg mL –1 . The ability of Bifidobacterium infantis CRL1395 to bind SBA on the bacterial envelope was confirmed, and prevention of IEC cytotoxicity by lectin removal was demonstrated. Safety of B. infantis CRL1395, resistance to gastrointestinal stress and adhesion were also determined. It was concluded that the early administration of B. infantis CRL1395 to chicks would effectively reduce the toxicity of SBA. Besides, it would favour the colonization of the gut with a beneficial microbiota.
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  • 24
    Publication Date: 2016-04-08
    Description: Fermentation-based production of butyric acid is robust and efficient. Modern catalytic technologies make it possible to convert butyric acid to important fine chemicals and biofuels. Here, current chemocatalytic and biocatalytic conversion methods are reviewed with a focus on upgrading butyric acid to 1-butanol or butyl-butyrate. Supported Ruthenium- and Platinum-based catalyst and lipase exhibit important activities which can pave the way for more sustainable process concepts for the production of green fuels and chemicals.
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  • 25
    Publication Date: 2016-04-15
    Description: Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner -based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP- based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A , thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum .
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  • 26
    Publication Date: 2016-04-15
    Description: Bacteria are becoming increasingly resistant to currently used antibiotics. At the same time, little progress has been made in discovering new antibacterial drugs to combat resistant organisms. History teaches us that ‘high tech’ target-based complex methods are not synonymous with success and a return to simple, systematic screening of natural products against bacteria from traditional and novel resources holds our greatest hope of success.
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  • 27
    Publication Date: 2016-04-15
    Description: The economic upturn of the past 200 years would not have been conceivable without fossil resources such as coal and oil. However, the fossil-based economy increasingly reaches its limits and displays contradictions. Bioeconomy, strategically combining economy and ecology willing to make biobased and sustainable growth possible, is promising to make a significant contribution towards solving these issues. In this context, microbial bioconversions are promising to support partially the increasing need for materials and fuels starting from fresh, preferably waste, biomass. Butanol is a very attractive molecule finding applications both as a chemical platform and as a fuel. Today it principally derives from petroleum, but it also represents the final product of microbial catabolic pathways. Because of the need to maximize yield, titer and productivity to make the production competitive and viable, the challenge is to transform a robustly regulated metabolic network into the principal cellular activity. However, this goal can only be accomplished by a profound understanding of the cellular physiology, survival strategy and sensing/signalling cascades. Here, we shortly review on the natural cellular pathways and circumstances that lead to n -butanol accumulation, its physiological consequences that might not match industrial needs and on possible solutions for circumventing these natural constraints.
    Keywords: Biotechnology & Synthetic Biology
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  • 28
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    Oxford University Press
    Publication Date: 2016-04-20
    Description: Butanol is a very interesting substance both for the chemical industry and as a biofuel. The classical distillation process for the removal of butanol is far too energy demanding, at a factor of 220% of the energy content of butanol. Alternative separation processes studied are hybrid processes of gas-stripping, liquid–liquid extraction and pervaporation with distillation and a novel adsorption/drying/desorption hybrid process. Compared with the energy content of butanol, the resulting energy demand for butanol separation and concentration of optimized hybrid processes is 11%–22% for pervaporation/distillation and 11%–17% for liquid–liquid extraction/distillation. For a novel adsorption/drying/desorption process, the energy demand is 9.4%. But all downstream process options need further proof of industrial applicability.
    Keywords: Biotechnology & Synthetic Biology
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  • 29
    Publication Date: 2016-04-20
    Description: Acetone–butanol–ethanol (ABE) fermentation is a metabolic process of clostridia that produces bio-based solvents including butanol. It is enabled by an underlying metabolic reaction network and modulated by cellular gene regulation and environmental cues. Mathematical modeling has served as a valuable strategy to facilitate the understanding, characterization and optimization of this process. In this review, we highlight recent advances in system-level, quantitative modeling of ABE fermentation. We begin with an overview of integrative processes underlying the fermentation. Next we survey modeling efforts including early simple models, models with a systematic metabolic description, and those incorporating metabolism through simple gene regulation. Particular focus is given to a recent system-level model that integrates the metabolic reactions, gene regulation and environmental cues. We conclude by discussing the remaining challenges and future directions towards predictive understanding of ABE fermentation.
    Keywords: Biotechnology & Synthetic Biology
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  • 30
    Publication Date: 2016-02-10
    Description: Cry proteins are pesticidal toxins produced by the bacterium Bacillus thuringiensis (Bt), which aggregate in sporulating cells to form a crystal. Except in a relatively few cases, these crystals are located outside the exosporium that surrounds the spore. Bt2-56 is a strain of Bt that has the relatively uncommon characteristic of locating its Cry protein-containing crystal within the exosporium, and in association with a long, multifiber filament. With the ultimate goal of both understanding and manipulating the localization of Cry proteins within the exosporium, we sought to identify the genes coding for the exosporium-localized Cry proteins in Bt2-56. Herein we show (i) that five cry-like genes are present in the genome of Bt2-56, (ii) that two pairs of these genes show organizational similarity to a relatively uncommon gene configuration that coexpress a cry gene along with a gene whose product aids crystal formation and (iii) that when one of these two gene pairs (cry21A-cdA) is expressed in an acrystalliferous strain of Bt, crystals are formed that localize within the exosporium. In Bt ssp. finitimus , the only other strain in which crystal localization has been studied, a Cry protein needed expression of two non-cry ORFs in order to localize within the exosporium, indicating that there are some mechanistic differences for crystal localization between Bt ssp. finitimus and Bt2-56.
    Keywords: Biotechnology & Synthetic Biology
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  • 31
    Publication Date: 2016-02-12
    Description: The commercialization of the n -butanol bioprocess is largely dependent on the price of feedstocks. Renewable cellulose appears to be an appealing feedstock. The microbial production of n -butanol still remains challenging because of the limited availability of intracellular NADH. To address this issue, an Escherichia coli strain carrying the clostridial CoA-dependent pathway was supplied with heterologous formate dehydrogenase. With the cellulose hydrolysate of rice straw, this single strain produced cellulosic biobutanol with a production yield at 35% of the theoretical and a productivity of 0.093 g L –1  h –1 . In an alternative method, the production involved a co-culture system consisting of two separate strains provided with the full CoA-dependent pathway. This system achieved a production yield and productivity reaching 62.8% of the theoretical and 0.163 g L –1  h –1 , respectively. The result indicates that the E. coli co-culture system is technically promising for the production of cellulosic biobutanol.
    Keywords: Biotechnology & Synthetic Biology
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  • 32
    Publication Date: 2016-11-09
    Description: Hepatitis E virus (HEV) is the pathogen causing hepatitis E (HE). It arouses global public health concern since it is a zoonotic disease. The objective of this letter is to report a cost-effective internal control prepared for monitoring procedures of HEV reverse transcriptase (RT)-PCR detection. A selected conserved HEV RNA fragment was integrated into the downstream of the truncated MS2 bacteriophage genome based on Armored RNA technology. The resulting MS2-HEV gene harbored by the pET-28b-MS2-HEV plasmid was transformed into E. coli BL21(DE3) for expression analysis by SDS-PAGE. The expression products were purified and concentrated by ultrasonication and ultrafiltration separation. The morphology and stability properties of the virus-like particles (VLPs) were evaluated by electron microscopy scanning and nuclease challenges, respectively. SDS-PAGE results showed that the constructed MS2-HEV gene expressed efficiently and the purity of the VLPs was highly consistent with the result in electron microscopy. Stability evaluation results demonstrated that the prepared VLPs exhibited strong resistance to DNase I and RNase A attacks and also performed long-lasting protection of coated HEV RNA for at least 4 months at –20°C. These data revealed that the prepared VLPs meet the basic requirements of use as internal control material in the HEV RNA amplification assay.
    Keywords: Biotechnology & Synthetic Biology
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  • 33
    Publication Date: 2016-09-08
    Description: Epigenetics is the study of heritable changes in gene expression without concomitant changes in DNA sequence. Due to its relevance in development, differentiation and human health, epigenetics has recently become an emerging area of science with regard to eukaryotic organisms and has shown enormous potential in synthetic biology. However, significant examples of epigenetic regulation in bacterial synthetic biology have not yet been reported. In the current study, we present the first model of such an epigenetic circuit. We termed the circuit the alternator circuit because parental cells carrying this circuit and their progeny alternate between distinct and heritable cellular fates without undergoing changes in genome sequence. Furthermore, we demonstrated that the alternator circuit exhibits hysteresis because its output depends not only on its present state but also on its previous states.
    Keywords: Biotechnology & Synthetic Biology
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  • 34
    Publication Date: 2016-10-12
    Description: Bacteriophages produce endolysins (peptidoglycan hydrolases) to lyse the host cell from within and release nascent bacteriophage particles. Recombinant endolysins can lyse Gram-positive bacteria when added exogenously. As a potential alternative antimicrobial, we cloned and expressed the enterococcal VD13 bacteriophage endolysin. VD13 endolysin has a CHAP catalytic domain with 92% identity with the bacteriophage IME-EF1 endolysin. The predicted size of VD13 endolysin is ~27 kDa as verified by SDS-PAGE. The VD13 endolysin lyses Enterococcus faecalis strains, but not E. faecium or other non-enterococci. VD13 endolysin has activity from pH 4 to pH 8, with peak activity at pH 5, and exhibits greater activity in the presence of calcium. Optimum activity at pH 5 occurs in the absence of NaCl. VD13 endolysin, in ammonium acetate (C 2 H 3 O 2 NH 4 ) calcium chloride (CaCl 2 ) buffer pH 5, is stimulated to higher activity upon heating at temperatures up to 65°C for 30 min, whereas activity is lost upon heating to 42°C, in pH 7 buffer.
    Keywords: Biotechnology & Synthetic Biology
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  • 35
    Publication Date: 2016-09-08
    Description: Cultivation in a bioreactor of immobilized deep-sea hydrothermal microbial community was tested in order to assess the stability and reactivity of this new system. A community composed of eight hydrothermal strains was entrapped in a polymer matrix that was used to inoculate a continuous culture in a gas-lift bioreactor. The continuous culture was performed for 41 days at successively 60°C, 55°C, 60°C, 85°C and 60°C, at pH 6.5, in anaerobic condition and constant dilution rate. Oxic stress and pH variations were tested at the beginning of the incubation. Despite these detrimental conditions, three strains including two strict anaerobes were maintained in the bioreactor. High cell concentrations (3 x 10 8 cells mL –1 ) and high ATP contents were measured in both liquid fractions and beads. Cloning-sequencing and qPCR revealed that Bacillus sp. dominated at the early stage, and was later replaced by Thermotoga maritima and Thermococcus sp. Acetate, formate and propionate concentrations varied simultaneously in the liquid fractions. These results demonstrate that these immobilized cells were reactive to culture conditions. They were protected inside the beads during the stress period and released in the liquid fraction when conditions were more favorable. This confirms the advantage of immobilization that highlights the resilience capacity of certain hydrothermal microorganisms after a stress period.
    Keywords: Biotechnology & Synthetic Biology
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  • 36
    Publication Date: 2016-10-12
    Description: To study the viability of a gyrA S83 stop mutation found in an Escherichia coli J53 ciprofloxacin-resistant strain (J53 CipR27), a pBR322 derivative was constructed with a TAG mutation in the bla gene knocking out ampicillin resistance. Ampicillin resistance was restored, suggesting that the strain contains tRNA suppressor activity able to suppress the UAG codon gyrA and allow viability. The method was applied to 22 unique clinical E. coli isolates, and all were found to have low-level suppressor activity.
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  • 37
    Publication Date: 2016-11-17
    Description: Enterotoxigenic Escherichia coli (ETEC) bacteria producing heat-stable toxin (STa) and/or heat-labile toxin (LT) are among top causes of children's diarrhea and travelers’ diarrhea. Currently no vaccines are available for ETEC associated diarrhea. A major challenge in developing ETEC vaccines is the inability to stimulate protective antibodies against the key STa toxin that is potently toxic and also poorly immunogenic. A recent study suggested toxoid fusion 3xSTa N12S -dmLT, which consists of a monomer LT toxoid (LT R192G/L211A ) and three copies of STa toxoid STa N12S , may represent an optimal immunogen inducing neutralizing antibodies against STa toxin [IAI 2014, 82(5):1823-32]. In this study, we immunized mice with this fusion protein following a different parenteral route and using different adjuvants to further characterize immunogenicity of this toxoid fusion. Data from this study showed that 3xSTa N12S -dmLT toxoid fusion induced neutralizing anti-STa antibodies in the mice following subcutaneous immunization, as effectively as in the mice under intraperitoneal route. Data also indicated that double mutant LT (dmLT) can be an effective adjuvant for this toxoid fusion in mice subcutaneous immunization. Results from this study affirmed that toxoid fusion 3xSTa N12S -dmLT induces neutralizing antibodies against STa toxin, suggesting this toxoid fusion is potentially a promising immunogen for ETEC vaccine development.
    Keywords: Biotechnology & Synthetic Biology
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  • 38
    Publication Date: 2016-12-23
    Description: Carocin D is a bacteriocin produced by Pectobacterium carotovorum subsp. carotovorum Pcc21. Carocin D inhibits the growth of P . carotovorum subsp. carotovorum and closely related strains. Pectobacterium carotovorum subsp. carotovorum is a causative bacterium for soft rot disease and leads to severe economic losses. Bacteriocins recognize and interact with a specific membrane protein of target bacteria as a receptor. To identify the receptor responsible for carocin D recognition, mutants that underwent a phenotypic change from carocin D sensitivity to carocin D insensitivity were screened. Based on Tn 5 insertions, carocin D sensitivity was dependent on expression of the outer membrane protein OmpF. The insensitivity of the mutant (Pcc3MR) to carocin D was complemented with ompF from carocin D-sensitive strains, not from carocin D-resistant strains. The selectivity between sensitive and resistant strains could be attributed to variation in OmpFs in the cell-surface-exposed regions. Based on sequence analysis and complementation assays, it appears that carocin D uses OmpF as a receptor and is translocated by the TonB system. According to previously reported translocation mechanisms of colicins, OmpF works along with the TolA system rather than the TonB system. Therefore, the current findings suggest that carocin D is imported by a unique colicin-like bacteriocin translocation system.
    Keywords: Biotechnology & Synthetic Biology
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  • 39
    Publication Date: 2016-11-17
    Description: Pseudomonas putida KT2440 is a saprophytic and generally recognized as safe microorganism that plays important roles in the biodegradation and production of value-added chemicals. Chromosomal gene deletion of P. putida KT2440 usually involves time-consuming gene cloning, conjugal transfer and counterselection. Recently, we developed a P. putida KT2440 markerless gene deletion method based on recombineering and Cre/ loxP site-specific recombination. PCR-based Red recombineering circumvents the tedious cloning steps and is more amenable to high-throughput manipulation. Here we report an improved scarless gene deletion strategy based on recombineering and intron-encoded homing endonuclease I-SceI-mediated double-strand break repair. Sixteen drug exporter gene(s) were deleted and the minimal inhibition concentrations of the mutants to a variety of antibiotics were determined. The robustness of the procedure was also demonstrated by sequential deletion of five large genomic regions. Up to 59% recombination efficiency was achieved for a 54.8 kb deletion, and the efficiency of RecA-mediated double-strand break repair, which was boosted by Red recombinase, was nearly 100%. The strain with a 3.76% genome reduction showed an improved growth rate and transformation efficiency. The straightforward, time-saving and highly efficient scarless deletion approach has the potential to facilitate the genetic study, and biotechnological and environmental applications of P. putida KT2440.
    Keywords: Biotechnology & Synthetic Biology
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  • 40
    Publication Date: 2016-12-16
    Description: Human health has been seriously endangered by highly prevalent salmonellosis and multidrug-resistant Salmonella strains. Current vaccines suffer from variable immune-protective effects, so more effective ones are needed to control Salmonella infection . Bacterial ghosts have been produced by the expression of lysis gene E from bacteriophage PhiX174 and can be filled with considerable exogenous substances such as DNA or drugs as a novel platform. In this study, Salmonella enteritidis (SE) ghosts were developed and loaded with Neisseria gonorrhoeae porin B (porB) to construct a novel inactive vaccine. Our new studies show that SE ghosts loaded with porB displayed increased production of pro-inflammatory cytokines (IL-1β, IL-6, IL-10 and IL-12p70) in bone marrow-derived dendritic cells (BMDCs), and elicited significantly higher specific systemic and mucosal immune responses to Salmonella than SE ghosts alone. In addition, the novel porB-loaded ghosts conferred higher protective effects on virulent Salmonella challenge. For the first time, we demonstrate that N. gonorrhoeae porB, as a novel adjuvant, can increase the immunogenicity of SE ghosts. Our studies suggested that Salmonella enteritidis ghosts loaded with Neisseria gonorrhoeae porin B might be a useful mucosal Salmonella vaccine candidate for practical use in the future.
    Keywords: Biotechnology & Synthetic Biology
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  • 41
    Publication Date: 2016-12-12
    Description: The food-grade bacterium Lactococcus lactis is increasingly used for heterologous protein expression in therapeutic and industrial applications. The ability of L. lactis to secrete biologically active cytokines may be used for the generation of therapeutic cytokines. Interleukin (IL)-18 enhances the immune response, especially on mucosal surfaces, emphasizing its therapeutic potential. However, it is produced as an inactive precursor and has to be enzymatically cleaved for maturation. We genetically manipulated L. lactis to secrete murine IL-18. The mature murine IL-18 gene was inserted downstream of a nisin promoter in pNZ8149 plasmid and the construct was used to transform L. lactis NZ3900 . The transformants were selected on Elliker agar and confirmed by restriction enzyme digestion and sequencing. The expression and secretion of IL-18 protein was verified by SDS-PAGE, western blotting and ELISA. The biological activity of recombinant IL-18 was determined by its ability to induce interferon (IFN)- production in L. lactis co-cultured with murine splenic T cells. The amounts of IL-18 in bacterial lysates and supernatants were 3–4 μg mL –1 and 0.6–0.7 ng mL –1 , respectively. The successfully generated L. lactis strain that expressed biologically active murine IL-18 can be used to evaluate the possible therapeutic effects of IL-18 on mucosal surfaces.
    Keywords: Biotechnology & Synthetic Biology
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