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  • Production, purification and characterization of recombinant human R-spondin1 (RSPO1) protein stably expressed in human HEK293 cells
    BMC Biotechnol. (IF 2.303) Pub Date : 2020-01-20
    Gabriel Levin; Bruna Andrade Aguiar Koga; Gustavo Gross Belchior; Ana Claudia Oliveira Carreira; Mari Cleide Sogayar

    The R-Spondin proteins comprise a family of secreted proteins, known for their important roles in cell proliferation, differentiation and death, by inducing the Wnt pathway. Several studies have demonstrated the importance of RSPOs in regulation of a number of tissue-specific processes, namely: bone formation, skeletal muscle tissue development, proliferation of pancreatic β-cells and intestinal stem cells and even cancer. RSPO1 stands out among RSPOs molecules with respect to its potential therapeutic use, especially in the Regenerative Medicine field, due to its mitogenic activity in stem cells. Here, we generated a recombinant human RSPO1 (rhRSPO1) using the HEK293 cell line, obtaining a purified, characterized and biologically active protein product to be used in Cell Therapy. The hRSPO1 coding sequence was synthesized and subcloned into a mammalian cell expression vector. HEK293 cells were stably co-transfected with the recombinant expression vector containing the hRSPO1 coding sequence and a hygromycin resistance plasmid, selected for hygror and subjected to cell clones isolation. rhRSPO1 was obtained, in the absence of serum, from culture supernatants of transfected HEK293 cells and purified using a novel purification strategy, involving two sequential chromatographic steps, namely: heparin affinity chromatography, followed by a molecular exclusion chromatography, designed to yield a high purity product. The purified protein was characterized by Western blotting, mass spectrometry and in vitro (C2C12 cells) and in vivo (BALB/c mice) biological activity assays, confirming the structural integrity and biological efficacy of this human cell expression system. Furthermore, rhRSPO1 glycosylation analysis allowed us to describe, for the first time, the glycan composition of this oligosaccharide chain, confirming the presence of an N-glycosylation in residue Asn137 of the polypeptide chain, as previously described. In addition, this analysis revealing the presence of glycan structures such as terminal sialic acid, N-acetylglucosamine and/or galactose. Therefore, a stable platform for the production and purification of recombinant hRSPO1 from HEK293 cells was generated, leading to the production of a purified, fully characterized and biologically active protein product to be applied in Tissue Engineering.

    更新日期:2020-01-21
  • Auxotrophic mutations of Trichophyton rubrum created by in vitro synthesized Cas9 ribonucleoprotein
    BMC Biotechnol. (IF 2.303) Pub Date : 2020-01-20
    Oliver Blechert; Huan Mei; Xiaohui Zang; Hailin Zheng; Guanzhao Liang; Weida Liu

    Trichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80–90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum. The gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method. We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing. We developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species.

    更新日期:2020-01-21
  • A self-aggregating peptide: implications for the development of thermostable vaccine candidates
    BMC Biotechnol. (IF 2.303) Pub Date : 2020-01-21
    Adolfo Cruz-Reséndiz; Jesús Zepeda-Cervantes; Alicia Sampieri; Carlos Bastián-Eugenio; Gonzalo Acero; J. Iván Sánchez-Betancourt; Goar Gevorkian; Luis Vaca

    The use of biomaterials has been expanded to improve the characteristics of vaccines. Recently we have identified that the peptide PH(1–110) from polyhedrin self-aggregates and incorporates foreign proteins to form particles. We have proposed that this peptide can be used as an antigen carrying system for vaccines. However, the immune response generated by the antigen fused to the peptide has not been fully characterized. In addition, the adjuvant effect and thermostability of the particles has not been evaluated. In the present study we demonstrate the use of a system developed to generate nano and microparticles carrying as a fusion protein peptides or proteins of interest to be used as vaccines. These particles are purified easily by centrifugation. Immunization of animals with the particles in the absence of adjuvant result in a robust and long-lasting immune response. Proteins contained inside the particles are maintained for over 1 year at ambient temperature, preserving their immunological properties. The rapid and efficient production of the particles in addition to the robust immune response they generate position this system as an excellent method for the rapid response against emerging diseases. The thermostability conferred by the particle system facilitates the distribution of the vaccines in developing countries or areas with no electricity.

    更新日期:2020-01-21
  • Construction and characterization of a high-quality cDNA library of Cymbidium faberi suitable for yeast one- and two-hybrid assays
    BMC Biotechnol. (IF 2.303) Pub Date : 2020-01-16
    Yanqin Xu; Junjiang Zhou; Qingqing Liu; Kunpeng Li; Yin Zhou

    Cymbidium faberi is one of the oldest cultivars of oriental orchids, with an elegant flower fragrance. In order to investigate the molecular mechanism and the functions of related proteins in the methyl jasmonate (MeJA) signaling pathway, one of the main components of flower fragrance in C. faberi, yeast one- and two-hybrid three-frame cDNA libraries were constructed. In this study, a modified cDNA library used for yeast one- and two-hybrid screening was successfully constructed, with a recombinant efficiency of 95%. The lengths of inserted fragments ranged from 750~3000 bp, and the library capacity reached 6 × 109 CFU/ μg of cDNA insert, which was suitable for the requirements of subsequent screening. Finally, a homologous protein related with pathogenesis was screened out by the bait vector of CfbHLH36, which may participate in the MeJA signaling pathway. The yeast one- and two-hybrid library of C. faberi provides large amounts of useful information for the functional genomics research in C. faberi, and this method could also be applied to other plants to screen DNA-protein and protein-protein interactions.

    更新日期:2020-01-16
  • Intein-mediated recombinant expression of monomeric B22Asp desB30 insulin
    BMC Biotechnol. (IF 2.303) Pub Date : 2020-01-09
    Minmin Zhang; Yunlong Zhang; Bingnan Wu; Yanhao Peng; Altaf Ahmed Simair; Geoffery W. Siegel; Changrui Lu; Ting Chen

    Insulin controls hyperglycemia caused by diabetes, and virtually all treatments require exogenous insulin. However, the product’s extensive post-translational modifications have hindered the manufacture of recombinant insulin. Here we report a novel production method for a monomeric B22Asp desB30 insulin analog (B22D desB30 insulin). Its precursor, DPIP, is fused to an N-terminal chitin-binding domain and intein self-cleavage tag. The fusion protein is expressed and purified from E. coli and immobilized on chitin resins. DPIP is then released using an optimized pH shift and converted to mature insulin via trypsin digest. The resulting product appears monomeric, > 90% pure and devoid of any exogenous enzyme. Thus, biologically active insulin analog can be efficiently produced in bacteria and potentially applicable in the treatment of human diabetes.

    更新日期:2020-01-11
  • Screening of cellulolytic bacteria from rotten wood of Qinling (China) for biomass degradation and cloning of cellulases from Bacillus methylotrophicus
    BMC Biotechnol. (IF 2.303) Pub Date : 2020-01-07
    Lingling Ma; Yingying Lu; Hong Yan; Xin Wang; Yanglei Yi; Yuanyuan Shan; Bianfang Liu; Yuan Zhou; Xin Lü

    Cellulosic biomass degradation still needs to be paid more attentions as bioenergy is the most likely to replace fossil energy in the future, and more evaluable cellulolytic bacteria isolation will lay a foundation for this filed. Qinling Mountains have unique biodiversity, acting as promising source of cellulose-degrading bacteria exhibiting noteworthy properties. Therefore, the aim of this work was to find potential cellulolytic bacteria and verify the possibility of the cloning of cellulases from the selected powerful bacteria. In present study, 55 potential cellulolytic bacteria were screened and identified from the rotten wood of Qinling Mountains. Based on the investigation of cellulase activities and degradation effect on different cellulose substrates, Bacillus methylotrophicus 1EJ7, Bacillus subtilis 1AJ3 and Bacillus subtilis 3BJ4 were further applied to hydrolyze wheat straw, corn stover and switchgrass, and the results suggested that B. methylotrophicus 1EJ7 was the most preponderant bacterium, and which also indicated that Bacillus was the main cellulolytic bacteria in rotten wood. Furthermore, scanning electron microscopy (SEM) and X-ray diffraction analysis of micromorphology and crystallinity of wheat straw also verified the significant hydrolyzation. With ascertaining the target sequence of cellulase β-glucosidase (243 aa) and endoglucanase (499 aa) were successfully heterogeneously cloned and expressed from B. methylotrophicus 1EJ7, and which performed a good effect on cellulose degradation with enzyme activity of 1670.15 ± 18.94 U/mL and 0.130 ± 0.002 U/mL, respectively. In addition, based on analysis of amino acid sequence, it found that β-glucosidase were belonged to GH16 family, and endoglucanase was composed of GH5 family catalytic domain and a carbohydrate-binding module of CBM3 family. Based on the screening, identification and cellulose degradation effect evaluation of cellulolytic bacteria from rotten wood of Qinling Mountains, it found that Bacillus were the predominant species among the isolated strains, and B. methylotrophicus 1EJ7 performed best on cellulose degradation. Meanwhile, the β-glucosidase and endoglucanase were successfully cloned and expressed from B. methylotrophicus for the first time, which provided new materials of both strain and the recombinant enzymes for the study of cellulose degradation and its application in industry.

    更新日期:2020-01-07
  • Development and applications of a monoclonal antibody against caprine interferon-gamma
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-23
    Wen-Tao Ma; Qi Liu; Meng-Xia Ning; Yu-Xu Qi; Saad Rehman; De-Kun Chen

    Interferon-gamma (IFN-γ) is an important mediator of type I immune response and has antiviral, immunoregulatory and anti-tumor properties, plays a wide range of roles in inflammation and autoimmune diseases. The aim of this study was to obtain monoclonal antibody (mAb) against caprine IFN-γ by immunizing of BALB/c mice with the purified rIFN-γ. Recombinant caprine IFN-γ was expressed in Escherichia coli strain BL21 (DE3) and monoclonal antibodies against caprine IFN-γ were produced by immunizing of BALB/c mice with rIFN-γ. One hybridoma secreting mAb was screened by enzyme-linked immunosorbent assay (ELISA) which was designated as 2C. MAb secreted by this cell line were analyzed through ELISA, western blot and application of the mAb was evaluated by immunofluorescence analysis using goat lip tissues infected with Orf virus. ELISA analysis revealed that mAb 2C can specifically recognize rIFN-γ protein and culture supernatant of goat peripheral blood mononuclear cells (PBMCs) stimulated by concanavalin A (Con A) but cannot recognize the fusion tag protein of pET-32a. Western blot analysis showed that mAb 2C can specifically react with the purified 34.9 kDa rIFN-γ protein but does not react with the fusion tag protein of pET-32a. Immunofluorescence results demonstrated that mAb 2C can detect IFN-γ secreted in histopathological sites of goats infected with Orf virus. A caprine IFN-γ-specific mAb was successfully developed in this study. Further analyses showed that the mAb can be used to detect IFN-γ expression level during contagious ecthyma in goats.

    更新日期:2019-12-23
  • The next generation of biopanning: next gen sequencing improves analysis of bacterial display libraries
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-21
    Sarah D. Stellwagen; Deborah A. Sarkes; Bryn L. Adams; Mia A. Hunt; Rebecca L. Renberg; Margaret M. Hurley; Dimitra N. Stratis-Cullum

    Bacterial surface display libraries are a popular tool for novel ligand discovery due to their ease of manipulation and rapid growth rates. These libraries typically express a scaffold protein embedded within the outer membrane with a short, surface-exposed peptide that is either terminal or is incorporated into an outer loop, and can therefore interact with and bind to substrates of interest. In this study, we employed a novel bacterial peptide display library which incorporates short 15-mer peptides on the surface of E. coli, co-expressed with the inducible red fluorescent protein DsRed in the cytosol, to investigate population diversity over two rounds of biopanning. The naive library was used in panning trials to select for binding affinity against 3D printing plastic coupons made from polylactic acid (PLA). Resulting libraries were then deep-sequenced using next generation sequencing (NGS) to investigate selection and diversity. We demonstrated enrichment for PLA binding versus a sapphire control surface, analyzed population composition, and compared sorting rounds using a binding assay and fluorescence microscopy. The capability to produce and describe display libraries through NGS across rounds of selection allows a deeper understanding of population dynamics that can be better directed towards peptide discovery.

    更新日期:2019-12-21
  • Optimized production of a biologically active Clostridium perfringens glycosyl hydrolase phage endolysin PlyCP41 in plants using virus-based systemic expression
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-21
    Rosemarie W. Hammond; Steven M. Swift; Juli A. Foster-Frey; Natalia Y. Kovalskaya; David M. Donovan

    Clostridium perfringens, a gram-positive, anaerobic, rod-shaped bacterium, is the third leading cause of human foodborne bacterial disease and a cause of necrotic enteritis in poultry. It is controlled using antibiotics, widespread use of which may lead to development of drug-resistant bacteria. Bacteriophage-encoded endolysins that degrade peptidoglycans in the bacterial cell wall are potential replacements for antibiotics. Phage endolysins have been identified that exhibit antibacterial activities against several Clostridium strains. An Escherichia coli codon-optimized gene encoding the glycosyl hydrolase endolysin (PlyCP41) containing a polyhistidine tag was expressed in E. coli. In addition, The E. coli optimized endolysin gene was engineered for expression in plants (PlyCP41p) and a plant codon-optimized gene (PlyCP41pc), both containing a polyhistidine tag, were expressed in Nicotiana benthamiana plants using a potato virus X (PVX)-based transient expression vector. PlyCP41p accumulated to ~ 1% total soluble protein (100μg/gm f. wt. leaf tissue) without any obvious toxic effects on plant cells, and both the purified protein and plant sap containing the protein lysed C. perfringens strain Cp39 in a plate lysis assay. Optimal systemic expression of PlyCP41p was achieved at 2 weeks-post-infection. PlyCP41pc did not accumulate to higher levels than PlyCP41p in infected tissue. We demonstrated that functionally active bacteriophage PlyCP41 endolysin can be produced in systemically infected plant tissue with potential for use of crude plant sap as an effective antimicrobial agent against C. perfringens.

    更新日期:2019-12-21
  • Fecal DNA isolation and degradation in clam Cyclina sinensis: noninvasive DNA isolation for conservation and genetic assessment
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-19
    Min Zhang; Min Wei; Zhiguo Dong; Haibao Duan; Shuang Mao; Senlei Feng; Wenqian Li; Zepeng Sun; Jiawei Li; Kanglu Yan; Hao Liu; Xueping Meng; Hongxing Ge

    To avoid destructive sampling for conservation and genetic assessment, we isolated the DNA of clam Cyclina sinensis from their feces. DNA electrophoresis and PCR amplification were used to determine the quality of fecal DNA. And we analyzed the effects of different conditions on the degradation of feces and fecal DNA. The clear fecal DNA bands were detected by electrophoresis, and PCR amplification using clam fecal DNA as template was effective and reliable, suggesting that clam feces can be used as an ideal material for noninvasive DNA isolation. In addition, by analyzing the effects of different environmental temperatures and soaking times on the degradation of feces and fecal DNA, we found that the optimum temperature was 4 °C. In 15 days, the feces maintained good texture, and the quality of fecal DNA was good. At 28 °C, the feces degraded in 5 days, and the quality of fecal DNA was poor. The clam feces can be used as an ideal material for noninvasive DNA isolation. Moreover, the quality of fecal DNA is negatively correlated with environmental temperature and soaking time.

    更新日期:2019-12-20
  • The effect of diet and radiation on the bacterial symbiome of the melon fly, Zeugodacus cucurbitae (Coquillett)
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-18
    Elias D. Asimakis; Mahfuza Khan; Panagiota Stathopoulou; Carlos Caceres; Kostas Bourtzis; George Tsiamis

    Symbiotic bacteria contribute to a multitude of important biological functions such as nutrition and reproduction and affect multiple physiological factors like fitness and longevity in their insect hosts. The melon fly, Zeugodacus cucurbitae (Coquillett), is an important agricultural pest that affects a variety of cultivated plants belonging mostly to the Cucurbitaceae family. It is considered invasive and widespread in many parts of the world. Several approaches are currently being considered for the management of its populations including the environmentally friendly and effective sterile insect technique (SIT), as a component of an integrated pest management (IPM) strategy. In the present study, we examined the effect of diet and radiation on the bacterial symbiome of Z. cucurbitae flies with the use of Next Generation Sequencing technologies. Melon flies were reared on two diets at the larval stage, an artificial bran-based diet and on sweet gourd, which affected significantly the development of the bacterial profiles. Significant differentiation was also observed based on gender. The effect of radiation was mostly diet dependent, with irradiated melon flies reared on the bran diet exhibiting a significant reduction in species diversity and richness compared to their non-irradiated controls. Changes in the bacterial symbiome of the irradiated melon flies included a drastic reduction in the number of sequences affiliated with members of Citrobacter, Raoultella, and Enterobacteriaceae. At the same time, an increase was observed for members of Enterobacter, Providencia and Morganella. Interestingly, the irradiated male melon flies reared on sweet gourd showed a clear differentiation compared to their non-irradiated controls, namely a significant reduction in species richness and minor differences in the relative abundance for members of Enterobacter and Providencia. The two diets in conjunction with the irradiation affected significantly the formation of the bacterial symbiome. Melon flies reared on the bran-based artificial diet displayed significant changes in the bacterial symbiome upon irradiation, in all aspects, including species richness, diversity and composition. When reared on sweet gourd, significant changes occurred to male samples due to radiation, only in terms of species richness.

    更新日期:2019-12-18
  • The oesophageal diverticulum of Dirioxa pornia studied through micro-CT scan, dissection and SEM studies
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-18
    Kala Bhandari; Peter Crisp; Michael A. Keller

    Dirioxa pornia (Diptera, Tephritidae) (Island fly) is an Australian native species related to a number of pestiferous fruit flies but, unlike many of the pest species, has not been studied extensively due to its non-pest status. However, due to D. pornia’s apparent reliance on the bacteria for survival it is an ideal species to undertake studies into interaction between Tephritid species and bacteria associated with the intestinal tract. The oesophageal diverticulum, which is a blind-ended protrusion of the oesophagus, has been studied, described and characterised in many other Tephritid species. Unlike many other species where the oesophageal diverticulum has been observed the organ was only observed in male D. pornia. It is speculated that this sexual dimorphism the organ may be the primary location to host beneficial bacteria in the involved in the production of the nuptial gift and the mating success of this Tephritid species. In case of D. pornia, however, no study on any area of the digestive system has been conducted. This study was conducted to locate and characterize the oesophageal diverticulum in D. pornia. A virtual dissection of the alimentary tract was made through micro-computer tomography studies. These studies were followed by dissection and scanning microscopy studies to elucidate the presence of bacteria. The oesophageal diverticulum of D. pornia is part of the foregut and distends from the oesophagus within the head of the fly. The shape of the oesophageal diverticulum corresponds with the Ceratitis type. Scanning microscopy studies of the oesophageal diverticulum show rod-shaped bacterial cells residing along with yeast cells in the lumen. The organ was only observed in male specimens. This study classifies the oesophageal diverticulum of D. pornia under the “Ceratitis type” of oesophageal diverticula in Tephritid species. The study also proves that micro-CT scanning is possible to locate soft tissues in Tephritid species and the Avizo® Fire software can be successfully used to visualize 3 dimensional (3D) images from x-rays. The methods used in this experiment can be used in future studies for visualising soft tissues of adult Tephritid species through micro tomography. There is sexual dimorphism with the organ only found in males. Finally this study shows that bacteria are present in the oesophageal diverticulum of D. pornia.

    更新日期:2019-12-18
  • Biochemical and nutritional characterization of the medfly gut symbiont Enterobacter sp. AA26 for its use as probiotics in sterile insect technique applications
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-18
    Konstantinos Azis; Ioanna Zerva; Paraschos Melidis; Carlos Caceres; Kostas Bourtzis; Spyridon Ntougias

    Enterobacter sp. AA26 was recently isolated from the midgut of Ceratitis capitata (Wiedemann) and it was shown to have positive effects in rearing efficiency when used as larval probiotics. In this study, biomass production was carried out in bench-scale bioreactors to elucidate the biokinetic properties of Enterobacter sp. AA26 and its nutritional value. Strain AA26 is a psychrotolerant, halotolerant, facultatively anaerobic bacterium with broad pH range for growth (pH 4 to 10.2), which possessed the typical biochemical profile of Enterobacter spp. The specific oxygen uptake rate (SOUR) was calculated as 63.2 ± 1.26 and 121 ± 1.73 mg O2 g− 1 VSS h− 1, with the yield coefficients in acetate and glucose being equal to 0.62 ± 0.03 and 0.67 ± 0.003 g biomass produced/g substrate consumed, respectively. The maximum specific growth rate (μmax) of strain AA26 grown in fill-and-draw bioreactors at 20 °C and 35 °C was 0.035 and 0.069 h− 1, respectively. Strain AA26 grew effectively in agro-industrial wastewaters, i.e. cheese whey wastewater (CWW), as alternative substrate for replacing yeast-based media. Biomass of strain AA26 could provide all the essential amino acids and vitamins for the artificial rearing of C. capitata. Greater intracellular α- and β-glucosidase activities were observed during growth of strain AA26 in CWW than in yeast-based substrate, although the opposite pattern was observed for the respective extracellular activities (p < 0.01). Low protease activity was exhibited in cells grown in yeast-based medium, while no lipase activities were detected. The ability of strain AA26 to grow in agro-industrial wastes and to provide all the essential nutrients can minimize the cost of commercial media used for mass rearing and large scale sterile insect technique applications.

    更新日期:2019-12-18
  • Olive fruit fly rearing procedures affect the vertical transmission of the bacterial symbiont Candidatus Erwinia dacicola
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-18
    Patrizia Sacchetti; Roberta Pastorelli; Gaia Bigiotti; Roberto Guidi; Sara Ruschioni; Carlo Viti; Antonio Belcari

    The symbiosis between the olive fruit fly, Bactrocera oleae, and Candidatus Erwinia dacicola has been demonstrated as essential for the fly’s larval development and adult physiology. The mass rearing of the olive fruit fly has been hindered by several issues, including problems which could be related to the lack of the symbiont, presumably due to preservatives and antibiotics currently used during rearing under laboratory conditions. To better understand the mechanisms underlying symbiont removal or loss during the rearing of lab colonies of the olive fruit fly, we performed experiments that focused on bacterial transfer from wild female flies to their eggs. In this research, eggs laid by wild females were treated with propionic acid solution, which is often used as an antifungal agent, a mixture of sodium hypochlorite and Triton X, or water (as a control). The presence of the bacterial symbiont on eggs was evaluated by real-time PCR and scanning electron microscopy. DGGE analysis showed a clear band with the same migration behavior present in all DGGE profiles but with a decreasing intensity. Molecular analyses performed by real-time PCR showed a significant reduction in Ca. E. dacicola abundance in eggs treated with propionic acid solution or a mixture of sodium hypochlorite and Triton X compared to those treated with water. In addition, the removal of bacteria from the surfaces of treated eggs was highlighted by scanning electron microscopy. The results clearly indicate how the first phases of the colony-establishment process are important in maintaining the symbiont load in laboratory populations and suggest that the use of products with antimicrobial activity should be avoided. The results also suggest that alternative rearing procedures for the olive fruit fly should be investigated.

    更新日期:2019-12-18
  • The host fruit amplifies mutualistic interaction between Ceratitis capitata larvae and associated bacteria
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-18
    Doron Shalom Yishai Zaada; Michael Ben-Yosef; Boaz Yuval; Edouard Jurkevitch

    The Mediterranean fruit fly Ceratitis capitata is a major pest in horticulture. The development of fly larvae is mediated by bacterial decay in the fruit tissue. Despite the importance of bacteria on larval development, very little is known about the interaction between bacteria and larvae in their true ecological context. Understanding their relationship and inter-dependence in the host fruit is important for the development of new pest control interfaces to deal with this pest. We find no negative effects on egg hatch or larval development brought about by the bacterial isolates tested. The various symbionts inhabiting the fly’s digestive system differ in their degree of contribution to the development of fly larvae depending on the given host and their sensitivity to induced inhibition caused by female produced antimicrobial peptides. These differences were observed not only at the genus or species level but also between isolates of the same species. We demonstrate how the microbiota from the mother’s gut supports the development of larvae in the fruit host and show that larvae play a major role in spreading the bacterial contagion in the infected fruit itself. In addition, we present (for the first time) evidence for horizontal transfer of bacteria between larvae of different maternal origin that develop together in the same fruit. Larvae play a major role in the spread and shaping of the microbial population in the fruit. The transfer of bacteria between different individuals developing in the same fruit suggests that the infested fruit serves as a microbial hub for the amplification and spread of bacterial strains between individuals.

    更新日期:2019-12-18
  • Horizontal transfer and finalization of a reliable detection method for the olive fruit fly endosymbiont, Candidatus Erwinia dacicola
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-18
    Gaia Bigiotti; Roberta Pastorelli; Roberto Guidi; Antonio Belcari; Patrizia Sacchetti

    The olive fly, Bactrocera oleae, is the most important insect pest in olive production, causing economic damage to olive crops worldwide. In addition to extensive research on B. oleae control methods, scientists have devoted much effort in the last century to understanding olive fly endosymbiosis with a bacterium eventually identified as Candidatus Erwinia dacicola. This bacterium plays a relevant role in olive fly fitness. It is vertically transmitted, and it benefits both larvae and adults in wild populations; however, the endosymbiont is not present in lab colonies, probably due to the antibiotics and preservatives required for the preparation of artificial diets. Endosymbiont transfer from wild B. oleae populations to laboratory-reared ones allows olive fly mass-rearing, thus producing more competitive flies for future Sterile Insect Technique (SIT) applications. We tested the hypothesis that Ca. E. dacicola might be transmitted from wild, naturally symbiotic adults to laboratory-reared flies. Several trials have been performed with different contamination sources of Ca. E. dacicola, such as ripe olives and gelled water contaminated by wild flies, wax domes containing eggs laid by wild females, cages dirtied by faeces dropped by wild flies and matings between lab and wild adults. PCR-DGGE, performed with the primer set 63F-GC/518R, demonstrated that the transfer of the endosymbiont from wild flies to lab-reared ones occurred only in the case of cohabitation. Cohabitation of symbiotic wild flies and non-symbiotic lab flies allows the transfer of Ca. E. dacicola through adults. Moreover, PCR-DGGE performed with the primer set 63F-GC/518R was shown to be a consistent method for screening Ca. E. dacicola, also showing the potential to distinguish between the two haplotypes (htA and htB). This study represents the first successful attempt at horizontal transfer of Ca. E. dacicola and the first step in acquiring a better understanding of the endosymbiont physiology and its relationship with the olive fly. Our research also represents a starting point for the development of a laboratory symbiotic olive fly colony, improving perspectives for future applications of the Sterile Insect Technique.

    更新日期:2019-12-18
  • Potential of a fly gut microbiota incorporated gel-based larval diet for rearing Bactrocera dorsalis (Hendel)
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-18
    Mahfuza Khan; Kajla Seheli; Md. Abdul Bari; Nahida Sultana; Shakil Ahmed Khan; Khandokar Fahmida Sultana; Md. Anwar Hossain

    The Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is an important polyphagous pest of horticultural produce. The sterile insect technique (SIT) is a proven control method against many insect pests, including fruit flies, under area-wide pest management programs. High quality mass-rearing process and the cost-effective production of sterile target species are important for SIT. Irradiation is reported to cause severe damage to the symbiotic community structure in the mid gut of fruit fly species, impairing SIT success. However, studies have found that target-specific manipulation of insect gut bacteria can positively impact the overall fitness of SIT-specific insects. Twelve bacterial genera were isolated and identified from B. dorsalis eggs, third instars larval gut and adults gut. The bacterial genera were Acinetobacter, Alcaligenes, Citrobacter, Pseudomonas, Proteus, and Stenotrophomonas, belonging to the Enterobacteriaceae family. Larval diet enrichment with the selected bacterial isolate, Proteus sp. was found to improve adult emergence, percentage of male, and survival under stress. However, no significant changes were recorded in B. dorsalis egg hatching, pupal yield, pupal weight, duration of the larval stage, or flight ability. These findings support the hypothesis that gut bacterial isolates can be used in conjunction with SIT. The newly developed gel-based larval diet incorporated with Proteus sp. isolates can be used for large-scale mass rearing of B. dorsalis in the SIT program.

    更新日期:2019-12-18
  • A walk on the wild side: gut bacteria fed to mass-reared larvae of Queensland fruit fly [Bactrocera tryoni (Froggatt)] influence development
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-18
    Lucas Alexander Shuttleworth; Mohammed Abul Monjur Khan; Terrence Osborne; Damian Collins; Mukesh Srivastava; Olivia Louise Reynolds

    The Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera, Tephritidae) is the most significant insect pest of Australian horticulture. Bactrocera tryoni is controlled using a range of tools including the Sterile Insect Technique (SIT). Mass-rearing and irradiation of pupae in SIT can reduce the fitness and quality of the released sterile insects. Studies have also showed reduced microbial gut diversity in domesticated versus wild tephritids. Transmission electron microscopy confirmed the presence of the bacterial isolates in the mid-gut of mass-reared larvae, and plate counts from individual larval guts showed increased numbers of bacteria in supplemented larvae. Several developmental and fitness parameters were tested including larval development time (egg-hatch to pupation), pupal weight, emergence, flight ability, sex-ratio, and time to adult eclosion (egg-hatch to adult eclosion). Enterobacter sp. and Asaia sp. shortened larval development time, while this was delayed by Lactobacillus sp., Leuconostoc sp. and a blend of all four bacteria. The mean time from egg hatch to adult eclosion was significantly reduced by Leuconostoc sp. and the blend for males and females, indicating that the individual bacterium and consortium affect flies differently depending on the life stage (larval or pupal). There was no impact of bacterial supplemented larvae on pupal weight, emergence, flight ability, or sex ratio. Our findings show that bacteria fed to the larval stage of B. tryoni can impart fitness advantages, but the selection of probiotic strains (individual or a consortium) is key, as each have varying effects on the host. Bacteria added to the larval diet particularly Leuconostoc sp. and the blend have the capacity to reduce costs and increase the number of flies produced in mass-rearing facilities by reducing time to adult eclosion by 1.3 and 0.8 mean days for males, and 1.2 and 0.8 mean days for females.

    更新日期:2019-12-18
  • Medfly-Wolbachia symbiosis: genotype x genotype interactions determine host’s life history traits under mass rearing conditions
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-18
    Georgios A. Kyritsis; Antonios A. Augustinos; Ioannis Livadaras; Carlos Cáceres; Kostas Bourtzis; Nikos T. Papadopoulos

    Wolbachia pipientis is a widespread, obligatory intracellular and maternally inherited bacterium, that induces a wide range of reproductive alterations to its hosts. Cytoplasmic Incompatibility (CI) is causing embryonic lethality, the most common of them. Despite that Wolbachia-borne sterility has been proposed as an environmental friendly pest control method (Incompatible Insect Technique, IIT) since 1970s, the fact that Wolbachia modifies important fitness components of its hosts sets severe barriers to IIT implementation. Mass rearing of Mediterranean fruit fly, Ceratitis capitata (medfly), is highly optimized given that this pest is a model species regarding the implementation of another sterility based pest control method, the Sterile Insect Technique (SIT). We used the medfly-Wolbachia symbiotic association, as a model system, to study the effect of two different Wolbachia strains, on the life history traits of 2 C. capitata lines with different genomic background. Wolbachia effects are regulated by both C. capitata genetic background and the Wolbachia strain. Wolbachia infection reduces fertility rates in both C. capitata genetic backgrounds and shortens the pre-pupa developmental duration in the GSS strain. On the other hand, regardless of the strain of Wolbachia (wCer2, wCer4) infection does not affect either the sex ratio or the longevity of adults. wCer4 infection imposed a reduction in females’ fecundity but wCer2 did not. Male mating competitiveness, adults flight ability and longevity under water and food deprivation were affected by both the genetic background of medfly and the strain of Wolbachia (genotype by genotype interaction). Wolbachia infection could alter important life history traits of mass-reared C. capitata lines and therefore the response of each genotype on the Wolbachia infection should be considered toward ensuring the productivity of the Wolbachia-infected insects under mass-rearing conditions.

    更新日期:2019-12-18
  • Simultaneous hydrolysis with lipase and fermentation of rapeseed cake for iturin A production by Bacillus amyloliquefaciens CX-20
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-16
    Wenchao Chen; Xuan Li; Xuli Ma; Shouwen Chen; Yanping Kang; Minmin Yang; Fenghong Huang; Xia Wan

    Rapeseed cake (RSC), as the intermediate by-product of oil extraction from the seeds of Brassica napus, can be converted into rapeseed meal (RSM) by solvent extraction to remove oil. However, compared with RSM, RSC has been rarely used as a raw material for microbial fermentation, although both RSC and RSM are mainly composed of proteins, carbohydrates and minerals. In this study, we investigated the feasibility of using untreated low-cost RSC as nitrogen source to produce the valuable cyclic lipopeptide antibiotic iturin A using Bacillus amyloliquefaciens CX-20 in submerged fermentation. Especially, the effect of oil in RSC on iturin A production and the possibility of using lipases to improve the iturin A production were analyzed in batch fermentation. The maximum production of iturin A was 0.82 g/L at the optimal initial RSC and glucose concentrations of 90 and 60 g/L, respectively. When RSC was substituted with RSM as nitrogen source based on equal protein content, the final concentration of iturin A was improved to 0.95 g/L. The production of iturin A was further increased by the addition of different lipase concentrations from 0.1 to 5 U/mL into the RSC medium for simultaneous hydrolysis and fermentation. At the optimal lipase concentration of 0.5 U/mL, the maximal production of iturin A reached 1.14 g/L, which was 38.15% higher than that without any lipase supplement. Although rapeseed oil and lipase were firstly shown to have negative effects on iturin A production, and the effect would be greater if the concentration of either was increased, their respective negative effects were reduced when used together. Appropriate relative concentrations of lipase and rapeseed oil were demonstrated to support optimal iturin A production. And simultaneous hydrolysis with lipase and fermentation was an effective way to produce iturin A from RSC using B. amyloliquefaciens CX-20.

    更新日期:2019-12-17
  • Morphological analysis of Apolipoprotein E binding to Aβ Amyloid using a combination of Surface Plasmon Resonance, Immunogold Labeling and Scanning Electron Microscopy
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-11
    Tohidul Islam; Anna L. Gharibyan; Cheng Choo Lee; Anders Olofsson

    Immunogold labeling in combination with transmission electron microscopy analysis is a technique frequently used to correlate high-resolution morphology studies with detailed information regarding localization of specific antigens. Although powerful, the methodology has limitations and it is frequently difficult to acquire a stringent system where unspecific low-affinity interactions are removed prior to analysis. We here describe a combinatorial strategy where surface plasmon resonance and immunogold labeling are used followed by a direct analysis of the sensor-chip surface by scanning electron microscopy. Using this approach, we have probed the interaction between amyloid-β fibrils, associated to Alzheimer’s disease, and apolipoprotein E, a well-known ligand frequently found co-deposited to the fibrillar form of Aβ in vivo. The results display a lateral binding of ApoE along the amyloid fibrils and illustrates how the gold-beads represent a good reporter of the binding. This approach exposes a technique with generic features which enables both a quantitative and a morphological evaluation of a ligand-receptor based system. The methodology mediates an advantage compared to traditional immunogold labeling since all washing steps can be monitored and where a high stringency can be maintained throughout the experiment.

    更新日期:2019-12-11
  • Purification of the recombinant green fluorescent protein from tobacco plants using alcohol/salt aqueous two-phase system and hydrophobic interaction chromatography
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-09
    Jie Dong; Xiangzhen Ding; Sheng Wang

    The green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics. A cost-efficient upstream expression system and an inexpensive downstream purification process will meet the demands of the GFP protein with high-purity. The recombinant GFP was transiently expressed in an active form in agoinoculated Nicotiana benthamiana leaves by using Tobacco mosaic virus (TMV) RNA-based overexpression vector (TRBO). The yield of recombinant GFP was up to ~ 60% of total soluble proteins (TSP). Purification of recombinant GFP from the clarified lysate of N. benthaniana leaves was achieved by using an alcohol/salt aqueous two-phase system (ATPS) and following with a further hydrophobic interaction chromatography (HIC). The purification process takes only ~ 4 h and can recover 34.1% of the protein. The purity of purified GFP was more than 95% and there were no changes in its spectroscopic characteristics. The strategy described here combines the advantages of both the economy and efficiency of plant virus-based expression platform and the simplicity and rapidity of environmentally friendly alcohol/salt ATPS. It has a considerable potential for the development of a cost-efficient alternative for production of recombinant GFP.

    更新日期:2019-12-11
  • A new method for quantitative detection of Lactobacillus casei based on casx gene and its application
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-10
    Xiaoyang Pang; Ziyang Jia; Jing Lu; Shuwen Zhang; Cai Zhang; Min Zhang; Jiaping Lv

    The traditional method of bacterial identification based on 16S rRNA is a widely used and very effective detection method, but this method still has some deficiencies, especially in the identification of closely related strains. A high homology with little differences is mostly observed in the 16S sequence of closely related bacteria, which results in difficulty to distinguish them by 16S rRNA-based detection method. In order to develop a rapid and accurate method of bacterial identification, we studied the possibility of identifying bacteria with other characteristic fragments without the use of 16S rRNA as detection targets. We analyzed the potential of using cas (CRISPR-associated proteins) gene as a target for bacteria detection. We found that certain fragment located in the casx gene was species-specific and could be used as a specific target gene. Based on these fragments, we established a TaqMan MGB Real-time PCR method for detecting bacteria. We found that the method used in this study had the advantages of high sensitivity and good specificity. The casx gene-based method of bacterial identification could be used as a supplement to the conventional 16 s rRNA-based detection method. This method has an advantage over the 16 s rRNA-based detection method in distinguishing the genetic relationship between closely-related bacteria, such as subgroup bacteria, and can be used as a supplement to the 16 s rRNA-based detection method.

    更新日期:2019-12-11
  • Improvement and use of CRISPR/Cas9 to engineer a sperm-marking strain for the invasive fruit pest Drosophila suzukii
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-12-05
    Hassan M. M. Ahmed; Luisa Hildebrand; Ernst A. Wimmer

    The invasive fruit pest Drosophila suzukii was reported for the first time in Europe and the USA in 2008 and has spread since then. The adoption of type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) as a tool for genome manipulation provides new ways to develop novel biotechnologically-based pest control approaches. Stage or tissue-specifically expressed genes are of particular importance in the field of insect biotechnology. The enhancer/promoter of the spermatogenesis-specific beta-2-tubulin (β2t) gene was used to drive the expression of fluorescent proteins or effector molecules in testes of agricultural pests and disease vectors for sexing, monitoring, and reproductive biology studies. Here, we demonstrate an improvement to CRISPR/Cas-based genome editing in D. suzukii and establish a sperm-marking system. To improve genome editing, we isolated and tested the D. suzukii endogenous promoters of the small nuclear RNA gene U6 to drive the expression of a guide RNA and the Ds heat shock protein 70 promoter to express Cas9. For comparison, we used recombinant Cas9 protein and in vitro transcribed gRNA as a preformed ribonucleoprotein. We demonstrate the homology-dependent repair (HDR)-based genome editing efficiency by applying a previously established transgenic line that expresses DsRed ubiquitously as a target platform. In addition, we isolated the Ds_β2t gene and used its promoter to drive the expression of a red fluorescence protein in the sperm. A transgenic sperm-marking strain was then established by the improved HDR-based genome editing. The deployment of the endogenous promoters of the D. suzukii U6 and hsp70 genes to drive the expression of gRNA and Cas9, respectively, enabled the effective application of helper plasmid co-injections instead of preformed ribonucleoproteins used in previous reports for HDR-based genome editing. The sperm-marking system should help to monitor the success of pest control campaigns in the context of the Sterile Insect Technique and provides a tool for basic research in reproductive biology of this invasive pest. Furthermore, the promoter of the β2t gene can be used in developing novel transgenic pest control approaches and the CRISPR/Cas9 system as an additional tool for the modification of previously established transgenes.

    更新日期:2019-12-06
  • Tuning the performance of CAR T cell immunotherapies
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-11-29
    Noah H. Richardson; Jordan B. Luttrell; Jonathan S. Bryant; Damian Chamberlain; Saleem Khawaja; Indira Neeli; Marko Radic

    Simultaneous advances in gene editing, T cell engineering and biotechnology currently provide an opportunity for rapid progress in medicine. The approval of chimeric antigen receptor (CAR) T cell therapies by the US Food and Drug Administration (FDA) and the European Commission have generated substantial momentum for these first-in-class therapies to be used in patients with B cell malignancies. Considerable efforts focus on improved outcomes and reduced side effects of the newly approved therapies. Using innovative strategies, researchers aim to extend CAR T cell use to tackle difficulties inherent in solid tumors. Efforts are underway to broaden the applications of CAR T cells, and the strategy has been successful in chronic viral infections and preclinical models of autoimmunity. Research is in progress to generate “off-the-shelf” CAR T cells, an advance, which would greatly increase patient availability and reduce treatment cost. In this thematic review, we highlight advances that may help develop genetically engineered cells into a new category of medical therapies.

    更新日期:2019-11-30
  • LC-MSMS based screening of emerging pollutant degradation by different peroxidases
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-11-28
    Khadega A. Almaqdi; Rana Morsi; Bahia Alhayuti; Farah Alharthi; S. Salman Ashraf

    The presence of a wide range of bioactive organic pollutants in wastewater and municipal water sources is raising concerns about their potential effects on humans. Not surprisingly, various approaches are being explored that can efficiently degrade these persistent organic pollutants. Use of peroxidases has recently been recognized as a novel remediation approach that may have potential advantages over conventional degradation techniques. However, testing the abilities of different peroxidases to degrade diverse emerging pollutants is tedious and cumbersome. In the present study, we present a rapid and robust approach to easily test the degradability of 21 different emerging pollutants by five different peroxidases (soybean peroxidase, chloroperoxidase, lactoperoxidase, manganese peroxidase, and horseradish peroxidase) using an LC-MSMS approach. Furthermore, this approach was also used to examine the role of a redox mediator in these enzymatic degradation assays. Our results show that some of the organic pollutants can be easily degraded by all five of the peroxidases tested, whereas others are only degraded by a specific peroxidase (or when a redox mediator was present) and there are some that are completely resistant to degradation by any of the peroxidases tested (even in the presence of a redox mediator). The degradation of furosemide and trimethoprim by soybean peroxidase and chloroperoxidase, respectively, was investigated in detail by examining the transformation products generated during their degradation. Some of the products generated during enzymatic breakdown of these pollutants have been previously reported by others, however, we report many new transformation products. LC-MSMS approaches, like the one described here, can be used to rapidly evaluate the potential of different peroxidases (and redox requirements) to be used as bioremediation agents. Our preliminary result shows peroxidases hold tremendous potential for being used in a final wastewater treatment step.

    更新日期:2019-11-29
  • Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-11-27
    Innanurdiani Koko; Adelene Ai-Lian Song; Mas Jaffri Masarudin; Raha Abdul Rahim

    Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901–1 integrating system. The constructed integration system comprises of a lactococcal promoter (PnisA or P170), phage attachment site (attP) from bacteriophage TP901–1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP344 anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P170 promoter with USP45 signal peptide which gave very low signals in whole cell ELISA. The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.

    更新日期:2019-11-28
  • A simple method for in vitro preparation of natural killer cells from cord blood
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-11-21
    Yong Xu Mu; Yu Xia Zhao; Bing Yao Li; Hong Jing Bao; Hui Jiang; Xiao Lei Qi; Li Yun Bai; Yun Hong Wang; Zhi Jie Ma; Xiao Yun Wu

    Cord Blood (CB) has been considered a promising source of natural killer (NK) cells for cellular immunotherapy. However, it is difficult to expand the large numbers of highly pure NK cells from CB without cell sorting and feeder cells/multiple cytokines. In this study, we try to develop a simple, safe and economical method for ex vivo expansion and purification of NK cells from CB without cell sorting and feeder cells/multiple cytokines. The large numbers (mean: 1.59 × 1010) of highly pure (≥90%) NK cells from CB could be obtained through interleukin-2, group A streptococcus and zoledronate stimulation of mononuclear cells using the 21-day culture approach. When compared to resting NK cells, expanded NK cells were a higher expression of activating receptors CD16, NKG2D, NKp30, NKp44, NKp46 and activating markers CD62L and CD69, while the inhibitory receptors, CD158a and CD158b remained largely unchanged. In addition, these cells showed a higher concentration of IFN-γ, TNF-α and GM-CSF secretion and cytotoxicity to K562 cells and acute myeloid leukemia targets than resting NK cells. We develop a simple, safe and economical method to obtain high yield, purity, and functionality NK cells from CB without cell sorting and feeder cells/multiple cytokines.

    更新日期:2019-11-21
  • Production and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid protein
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-11-21
    Razieh Yazdani; Masoud Shams-Bakhsh; Afshin Hassani-Mehraban; Seyed Shahriar Arab; Nicolas Thelen; Marc Thiry; Jacques Crommen; Marianne Fillet; Nathalie Jacobs; Alain Brans; Anne-Catherine Servais

    Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated. The epitope sequence was genetically inserted in the αB-αB” domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure. The results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest.

    更新日期:2019-11-21
  • Identification and characterization of the GmRD26 soybean promoter in response to abiotic stresses: potential tool for biotechnological application
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-11-20
    Elinea O. Freitas; Bruno P. Melo; Isabela T. Lourenço-Tessutti; Fabrício B. M. Arraes; Regina M. Amorim; Maria E. Lisei-de-Sá; Julia A. Costa; Ana G. B. Leite; Muhammad Faheem; Márcio A. Ferreira; Carolina V. Morgante; Elizabeth P. B. Fontes; Maria F. Grossi-de-Sa

    Drought is one of the most harmful abiotic stresses for plants, leading to reduced productivity of several economically important crops and, consequently, considerable losses in the agricultural sector. When plants are exposed to stressful conditions, such as drought and high salinity, they modulate the expression of genes that lead to developmental, biochemical, and physiological changes, which help to overcome the deleterious effects of adverse circumstances. Thus, the search for new specific gene promoter sequences has proved to be a powerful biotechnological strategy to control the expression of key genes involved in water deprivation or multiple stress responses. This study aimed to identify and characterize the GmRD26 promoter (pGmRD26), which is involved in the regulation of plant responses to drought stress. The expression profile of the GmRD26 gene was investigated by qRT-PCR under normal and stress conditions in Williams 82, BR16 and Embrapa48 soybean-cultivars. Our data confirm that GmRD26 is induced under water deficit with different induction folds between analyzed cultivars, which display different genetic background and physiological behaviour under drought. The characterization of the GmRD26 promoter was performed under simulated stress conditions with abscisic acid (ABA), polyethylene glycol (PEG) and drought (air dry) on A. thaliana plants containing the complete construct of pGmRD26::GUS (2.054 bp) and two promoter modules, pGmRD26A::GUS (909 pb) and pGmRD26B::GUS (435 bp), controlling the expression of the β-glucuronidase (uidA) gene. Analysis of GUS activity has demonstrated that pGmRD26 and pGmRD26A induce strong reporter gene expression, as the pAtRD29 positive control promoter under ABA and PEG treatment. The full-length promoter pGmRD26 and the pGmRD26A module provides an improved uidA transcription capacity when compared with the other promoter module, especially in response to polyethylene glycol and drought treatments. These data indicate that pGmRD26A may become a promising biotechnological asset with potential use in the development of modified drought-tolerant plants or other plants designed for stress responses.

    更新日期:2019-11-20
  • Novel anti-CD38 humanized mAb SG003 possessed enhanced cytotoxicity in lymphoma than Daratumumab via antibody-dependent cell-mediated cytotoxicity
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-05-22
    Tao Yu; Chunxia Qiao; Ming Lv; Luqun Tang

    In vivo use of monoclonal antibodies has become routine clinical practice in the treatment of human cancer. CD38 is an attractive target, because it has double roles, as a receptor and an ectoenzyme. Daratumumab, an anti-CD38 antibody, is currently in the clinical trials for multiple myeloma. Here we obtained a humanized anti-CD38 antibody, SG003, using SDR-grafting method. SG003 possessed stronger antigen binding activity than Daratumumab, and its epitope was far from that of Daratumumab, an anti-CD38 antibody currently in the clinical trials for multiple myeloma; besides, SG003 showed enhanced antibody-dependent cell-mediated cytotoxicity function and in vivo inhibitory efficacy of tumor growth in xenograft mice model. SG003 seemed to be a good option to improve the curative effect of CD38-related cancers.

    更新日期:2019-11-18
  • A novel autolysis system for extracellular production and direct immobilization of a phospholipase D fused with cellulose binding domain
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-05-22
    Haiyang Zhang; Wenqin Chu; Jianan Sun; Zhen Liu; Wen-can Huang; Changhu Xue; Xiangzhao Mao

    Several types of phospholipases have been described in phospholipids modification. The majority of phospholipase D (PLD) superfamily members can catalyze two separate reactions: the hydrolysis of phospholipids to produce phosphatidic acid (PA) and the transphosphatidylation of phosphatidyl groups into various phosphatidyl alcohols to produce modified phospholipids. Transphosphatidylation is a useful biocatalytic method for the synthesis of functional phospholipids from lecithin or phosphatidylcholine (PC), which are both easily accessible. Different PLD coding genes have been cloned from various sources from viral, prokaryotic, and eukaryotic organisms. Despite the catalytic potential of PLD, their low productivity has hampered their practical applications, probably because PLD, which is highly toxic to the host cells, when transformation of the PLD genes into the host cells, degrade PLs in the cell membrane. In this study, we designed a novel two-step expression system to produce and secrete recombinant PLD in extracellular medium, cellulose-binding domains as an affinity fused with PLD for immobilization and purification proteins. The engineered BL21 (DE3) host strain, which harbored the final expression vector pET28a-PLD-CBD-araC-ESN, was induced by IPTG and L-arabinose, the cell density decreased rapidly over a 2 h period and the enzymes released into the extracellular medium accounts owned 81.75% hydrolytic activity. Scanning electron microscopy results showed that there were obvious structural changes on the cell surface. The extracellularly secreted PLD-CBD powder was used to catalyze the transphosphatidylation reaction synthesis of phosphatidylserine, 2.3 U enzymes reacted for 12 h, during which the conversion rate reached 99% with very few by-products being produced. When the fused protein PLD-CBD immobilized on microcrystalline cellulose, the enzymes can be cycle used five times with 26% conversion rate was preserved. This study introduced an effective method for use in the expression of recombinant proteins and their extracellular secretion that simplifies the steps of sonication and purification and demonstrates great potential in the industrial application of enzymes. Cellulose as the most abundant renewable biomass resources in nature, and the cost is low, used for PLD immobilization make it more simple, effective and sustainable.

    更新日期:2019-11-18
  • A high throughput method for total alcohol determination in fermentation broths
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-05-22
    Peng Zhang; Hao Hai; Dongxu Sun; Weihua Yuan; Weijie Liu; Ruru Ding; Mengting Teng; Lin Ma; Jun Tian; Caifa Chen

    The potassium dichromate oxidation method used in determination of alcohols in fermentation has two major disadvantages. This method cannot be used to determine alcohols in raw fermentation broth samples, which often contain various reducing sugars. The method is not environment friendly due to the carcinogenicity of Cr (VI) used. A new method for determination of reducing sugars and total alcohols in raw fermentation broths was developed. The fermentation broth was pretreated to remove proteins, polysaccharides, glycerol and organic acids. The colorimetric change from both total alcohols and reducing sugars by potassium permanganate oxidation was measured. The portion of colorimetric change from oxidation of reducing sugars was determined by DNS test and subtracted. The remaining portion of colorimetric change was then used to calculate the total alcohol concentration in the sample. Using this method, total alcohol concentration can be easily and accurately determined in both distilled samples and raw fermentation broth samples. It is fast and environmental friendly.

    更新日期:2019-11-18
  • Copy number determination of the gene for the human pancreatic polypeptide receptor NPY4R using read depth analysis and droplet digital PCR
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-06-04
    Kateryna Shebanits; Torsten Günther; Anna C. V. Johansson; Khurram Maqbool; Lars Feuk; Mattias Jakobsson; Dan Larhammar

    Copy number variation (CNV) plays an important role in human genetic diversity and has been associated with multiple complex disorders. Here we investigate a CNV on chromosome 10q11.22 that spans NPY4R, the gene for the appetite-regulating pancreatic polypeptide receptor Y4. This genomic region has been challenging to map due to multiple repeated elements and its precise organization has not yet been resolved. Previous studies using microarrays were interpreted to show that the most common copy number was 2 per genome. We have investigated 18 individuals from the 1000 Genomes project using the well-established method of read depth analysis and the new droplet digital PCR (ddPCR) method. We find that the most common copy number for NPY4R is 4. The estimated number of copies ranged from three to seven based on read depth analyses with Control-FREEC and CNVnator, and from four to seven based on ddPCR. We suggest that the difference between our results and those published previously can be explained by methodological differences such as reference gene choice, data normalization and method reliability. Three high-quality archaic human genomes (two Neanderthal and one Denisova) display four copies of the NPY4R gene indicating that a duplication occurred prior to the human-Neanderthal/Denisova split. We conclude that ddPCR is a sensitive and reliable method for CNV determination, that it can be used for read depth calibration in CNV studies based on already available whole-genome sequencing data, and that further investigation of NPY4R copy number variation and its consequences are necessary due to the role of Y4 receptor in food intake regulation.

    更新日期:2019-11-18
  • Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-06-04
    Hong Gao; Buvani Murugesan; Janina Hoßbach; Stephanie K. Evans; W. Marshall Stark; Margaret C. M. Smith

    Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify more integrating vectors, using phage int/attP loci, that would efficiently integrate site-specifically in the rare Actinomycete, Amycolatopsis marina DSM45569. Analysis of the genome of A. marina DSM45569 indicated the presence of attB-like sequences for TG1 and R4 integrases. The TG1 and R4 attBs were active in in vitro recombination assays with their cognate purified integrases and attP loci. Integrating vectors containing either the TG1 or R4 int/attP loci yielded exconjugants in conjugation assays from Escherichia coli to A. marina DSM45569. Site-specific recombination of the plasmids into the host TG1 or R4 attB sites was confirmed by sequencing. The homologous TG1 and R4 attB sites within the genus Amycolatopsis have been identified. The results indicate that vectors based on TG1 and R4 integrases could be widely applicable in this genus.

    更新日期:2019-11-18
  • Investigation of cell culture conditions for optimal foot-and-mouth disease virus production
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-06-07
    Veronika Dill; Aline Zimmer; Martin Beer; Michael Eschbaumer

    Foot-and-mouth disease is a highly contagious and economically devastating disease with endemic occurrence in many parts of the world. Vaccination is the method of choice to eradicate the disease and to limit the viral spread. The vaccine production process is based on mammalian cell culture, in which the viral yield varies in dependence of the composition of the culture media. For foot-and-mouth disease virus (FMDV), very little is known about the culture media components that are necessary to grow the virus to high titers in cell culture. This study examined the influence of increasing concentrations of glucose, glutamine, ammonium chloride and different cell densities on the yield of FMDV. While an excess of glucose or glutamine does not affect the viral yield, increasing cell density reduces the viral titer by a log10 step at a cell density of 3 × 106 cells/mL. This can be mitigated by performing a 100% media exchange before infection of the cells. The reasons for the diminished viral growth, if no complete media exchange has been performed prior to infection, remain unclear and further studies are necessary to investigate the causes more deeply. For now, the results argue for a vaccine production process with 100% media exchange to reliably obtain high viral titers.

    更新日期:2019-11-18
  • New α- and SIN γ-retrovectors for safe transduction and specific transgene expression in pancreatic β cell lines
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-06-17
    Olivier Albagli; Alicia Maugein; Lukas Huijbregts; Delphine Bredel; Géraldine Carlier; Patrick Martin; Raphaël Scharfmann

    Viral vectors are invaluable tools to transfer genes and/or regulatory sequences into differentiated cells such as pancreatic cells. To date, several kinds of viral vectors have been used to transduce different pancreatic cell types, including insulin-producing β cells. However, few studies have used vectors derived from « simple » retroviruses, such as avian α- or mouse γ-retroviruses, despite their high experimental convenience. Moreover, such vectors were never designed to specifically target transgene expression into β cells. We here describe two novel α- or SIN (Self-Inactivating) γ-retrovectors containing the RIP (Rat Insulin Promoter) as internal promoter. These two retrovectors are easily produced in standard BSL2 conditions, rapidly concentrated if needed, and harbor a large multiple cloning site. For the SIN γ-retrovector, either the VSV-G (pantropic) or the retroviral ecotropic (rodent specific) envelope was used. For the α-retrovector, we used the A type envelope, as its receptor, termed TVA, is only naturally present in avian cells and can efficiently be provided to mammalian β cells through either exogenous expression upon cDNA transfer or gesicle-mediated delivery of the protein. As expected, the transgenes cloned into the two RIP-containing retrovectors displayed a strong preferential expression in β over non-β cells compared to transgenes cloned in their non-RIP (CMV- or LTR-) regulated counterparts. We further show that RIP activity of both retrovectors mirrored fluctuations affecting endogenous INSULIN gene expression in human β cells. Finally, both α- and SIN γ-retrovectors were extremely poorly mobilized by the BXV1 xenotropic retrovirus, a common invader of human cells grown in immunodeficient mice, and, most notably, of human β cell lines. Our novel α- and SIN γ-retrovectors are safe and convenient tools to stably and specifically express transgene(s) in mammalian β cells. Moreover, they both reproduce some regulatory patterns affecting INSULIN gene expression. Thus, they provide a helpful tool to both study the genetic control of β cell function and monitor changes in their differentiation status.

    更新日期:2019-11-18
  • Development of a luciferase/luciferin cell proliferation (XenoLuc) assay for real-time measurements of Gfp-Luc2-modified cells in a co-culture system
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-06-14
    Sin-Yeang Teow; Kitson Liew; Mohd Firdaus Che Mat; Marini Marzuki; Norazlin Abdul Aziz; Tai-Lin Chu; Munirah Ahmad; Alan Soo-Beng Khoo

    In vitro modelling of cancer cells is becoming more complex due to prevailing evidence of intimate interactions between cancer cells and their surrounding stroma. A co-culture system which consists of more than one cell type is physiologically more relevant and thus, could serve as a useful model for various biological studies. An assay that specifically detects the phenotypic changes of cancer cells in a multi-cellular system is lacking for nasopharyngeal carcinoma (NPC). Here, we describe a luciferase/luciferin (XenoLuc) assay that could specifically measure changes in the proliferation of cancer cells in the co-culture system using two modified NPC patient-derived tumour xenograft (PDTXs) cells: Xeno284-gfp-luc2 and XenoB110-gfp-luc2. Through this assay, we are able to show that the growth of NPC xenograft cells in both two-dimensional (2D) and three-dimensional (3D) models was enhanced when co-cultured with normal human dermal fibroblasts (NHDFs). In addition, potential applications of this assay in in vitro drug or inhibitor screening experiments are also illustrated. XenoLuc assay is specific, sensitive, rapid and cost-effective for measuring the growth of luciferase-expressing cells in a co- or multiple-culture system. This assay may also be adapted for tumour microenvironment studies as well as drug screening experiments in more complex 3D co-culture systems.

    更新日期:2019-11-18
  • Improved CRISPR/Cas9 gene editing by fluorescence activated cell sorting of green fluorescence protein tagged protoplasts
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-06-17
    Bent Larsen Petersen; Svenning Rune Möller; Jozef Mravec; Bodil Jørgensen; Mikkel Christensen; Ying Liu; Hans H. Wandall; Eric Paul Bennett; Zhang Yang

    CRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand. In this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3–5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations. FACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.

    更新日期:2019-11-18
  • Engineered Pichia pastoris production of fusaruside, a selective immunomodulator
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-06-17
    Yuan Tian; Yanling Li; Fengchun Zhao; Chao Meng

    Fusaruside is an immunomodulatory fungal sphingolipid which has medical potentials for treating colitis and liver injury, but its poor natural abundance limits its further study. In this study, we described a synthetic biology approach for fusaruside production by engineered Pichia pastoris that was based on polycistronic expression. Two fusaruside biosynthesis genes (Δ3(E)-sd and Δ10(E)-sd), were introduced into P. pastoris to obtain fusaruside producing strain FUS2. To further enhance the yield of fusaruside, three relevant biosynthetic genes (Δ3(E)-sd, Δ10(E)-sd and gcs) were subsequently introduced into P. pastoris to obtain FUS3. All of the biosynthetic genes were successfully co-expressed in FUS2 and FUS3. Compared to that produced by FUS2, fusaruside achieved from FUS3 were slightly increased. In addition, the culture conditions including pH, temperature and methanol concentration were optimized to improve the fusaruside production level. Here a novel P. pastoris fusaruside production system was developed by introducing the biosynthetic genes linked by 2A peptide gene sequences into a polycistronic expression construct, laying a foundation for further development and application of fusaruside.

    更新日期:2019-11-18
  • Mucosal delivery of Lactococcus lactis carrying an anti-TNF scFv expression vector ameliorates experimental colitis in mice
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-06-25
    Maria José Chiabai; Juliana Franco Almeida; Mariana Gabriela Dantas de Azevedo; Suelen Soares Fernandes; Vanessa Bastos Pereira; Raffael Júnio Araújo de Castro; Márcio Sousa Jerônimo; Isabel Garcia Sousa; Leonora Maciel de Souza Vianna; Anderson Miyoshi; Anamelia Lorenzetti Bocca; Andrea Queiroz Maranhão; Marcelo Macedo Brigido

    Anti-Tumor Necrosis Factor-alpha therapy has become clinically important for treating inflammatory bowel disease. However, the use of conventional immunotherapy requires a systemic exposure of patients and collateral side effects. Lactic acid bacteria have been shown to be effective as mucosal delivering system for cytokine and single domain antibodies, and it is amenable to clinical purposes. Therefore, lactic acid bacteria may function as vehicles for delivery of therapeutic antibodies molecules to the gastrointestinal tract restricting the pharmacological effect towards the gut. Here, we use the mucosal delivery of Lactococcus lactis carrying an anti-TNFα scFv expression plasmid on a DSS-induced colitis model in mice. Experimental colitis was induced with DSS administered in drinking water. L. lactis carrying the scFv expression vector was introduced by gavage. After four days of treatment, animals showed a significant improvement in histological score and disease activity index compared to those of untreated animals. Moreover, treated mice display IL-6, IL17A, IL1β, IL10 and FOXP3 mRNA levels similar to health control mice. Therefore, morphological and molecular markers suggest amelioration of the experimentally induced colitis. These results provide evidence for the use of this alternative system for delivering therapeutic biopharmaceuticals in loco for treating inflammatory bowel disease, paving the way for a novel low-cost and site-specific biotechnological route for the treatment of inflammatory disorders.

    更新日期:2019-11-18
  • Genome-wide response on phytosterol in 9-hydroxyandrostenedione-producing strain of Mycobacterium sp. VKM Ac-1817D
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-06-25
    Eugeny Y. Bragin; Victoria Y. Shtratnikova; Mikhail I. Schelkunov; Dmitry V. Dovbnya; Marina V. Donova

    Aerobic side chain degradation of phytosterols by actinobacteria is the basis for the industrial production of androstane steroids which are the starting materials for the synthesis of steroid hormones. A native strain of Mycobacterium sp. VKM Ac-1817D effectively produces 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) from phytosterol, but also is capable of slow steroid core degradation. However, the set of the genes with products that are involved in phytosterol oxidation, their organisation and regulation remain poorly understood. High-throughput sequencing of the global transcriptomes of the Mycobacterium sp. VKM Ac-1817D cultures grown with or without phytosterol was carried out. In the presence of phytosterol, the expression of 260 genes including those related to steroid catabolism pathways significantly increased. Two of the five genes encoding the oxygenase unit of 3-ketosteroid-9α-hydroxylase (kshA) were highly up-regulated in response to phytosterol (55- and 25-fold, respectively) as well as one of the two genes encoding its reductase subunit (kshB) (40-fold). Only one of the five putative genes encoding 3-ketosteroid-∆1-dehydrogenase (KstD_1) was up-regulated in the presence of phytosterol (61-fold), but several substitutions in the conservative positions of its product were revealed. Among the genes over-expressed in the presence of phytosterol, several dozen genes did not possess binding sites for the known regulatory factors of steroid catabolism. In the promoter regions of these genes, a regularly occurring palindromic motif was revealed. The orthologue of TetR-family transcription regulator gene Rv0767c of M. tuberculosis was identified in Mycobacterium sp. VKM Ac-1817D as G155_05115. High expression levels of the genes related to the sterol side chain degradation and steroid 9α-hydroxylation in combination with possible defects in KstD_1 may contribute to effective 9α-hydroxyandrost-4-ene-3,17-dione accumulation from phytosterol provided by this biotechnologically relevant strain. The TetR-family transcription regulator gene G155_05115 presumably associated with the regulation of steroid catabolism. The results are of significance for the improvement of biocatalytic features of the microbial strains for the steroid industry.

    更新日期:2019-11-18
  • VARSCOT: variant-aware detection and scoring enables sensitive and personalized off-target detection for CRISPR-Cas9
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-06-27
    Laurence O. W. Wilson; Sara Hetzel; Christopher Pockrandt; Knut Reinert; Denis C. Bauer

    Natural variations in a genome can drastically alter the CRISPR-Cas9 off-target landscape by creating or removing sites. Despite the resulting potential side-effects from such unaccounted for sites, current off-target detection pipelines are not equipped to include variant information. To address this, we developed VARiant-aware detection and SCoring of Off-Targets (VARSCOT). VARSCOT identifies only 0.6% of off-targets to be common between 4 individual genomes and the reference, with an average of 82% of off-targets unique to an individual. VARSCOT is the most sensitive detection method for off-targets, finding 40 to 70% more experimentally verified off-targets compared to other popular software tools and its machine learning model allows for CRISPR-Cas9 concentration aware off-target activity scoring. VARSCOT allows researchers to take genomic variation into account when designing individual or population-wide targeting strategies. VARSCOT is available from https://github.com/BauerLab/VARSCOT .

    更新日期:2019-11-18
  • Genome-wide sequencing and metabolic annotation of Pythium irregulare CBS 494.86: understanding Eicosapentaenoic acid production
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-06-28
    Bruna S. Fernandes; Oscar Dias; Gisela Costa; Antonio A. Kaupert Neto; Tiago F. C. Resende; Juliana V. C. Oliveira; Diego M. Riaño-Pachón; Marcelo Zaiat; José G. C. Pradella; Isabel Rocha

    Pythium irregulare is an oleaginous Oomycete able to accumulate large amounts of lipids, including Eicosapentaenoic acid (EPA). EPA is an important and expensive dietary supplement with a promising and very competitive market, which is dependent on fish-oil extraction. This has prompted several research groups to study biotechnological routes to obtain specific fatty acids rather than a mixture of various lipids. Moreover, microorganisms can use low cost carbon sources for lipid production, thus reducing production costs. Previous studies have highlighted the production of EPA by P. irregulare, exploiting diverse low cost carbon sources that are produced in large amounts, such as vinasse, glycerol, and food wastewater. However, there is still a lack of knowledge about its biosynthetic pathways, because no functional annotation of any Pythium sp. exists yet. The goal of this work was to identify key genes and pathways related to EPA biosynthesis, in P. irregulare CBS 494.86, by sequencing and performing an unprecedented annotation of its genome, considering the possibility of using wastewater as a carbon source. Genome sequencing provided 17,727 candidate genes, with 3809 of them associated with enzyme code and 945 with membrane transporter proteins. The functional annotation was compared with curated information of oleaginous organisms, understanding amino acids and fatty acids production, and consumption of carbon and nitrogen sources, present in the wastewater. The main features include the presence of genes related to the consumption of several sugars and candidate genes of unsaturated fatty acids production. The whole metabolic genome presented, which is an unprecedented reconstruction of P. irregulare CBS 494.86, shows its potential to produce value-added products, in special EPA, for food and pharmaceutical industries, moreover it infers metabolic capabilities of the microorganism by incorporating information obtained from literature and genomic data, supplying information of great importance to future work.

    更新日期:2019-11-18
  • Primary allogeneic mitochondrial mix (PAMM) transfer/transplant by MitoCeption to address damage in PBMCs caused by ultraviolet radiation
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-06-28
    Francisco Cabrera; Mayra Ortega; Francesca Velarde; Eliseo Parra; Stephany Gallardo; Diego Barba; Lina Soto; Gabriela Peña; Luis Alberto Pedroza; Christian Jorgensen; Maroun Khoury; Andrés Caicedo

    Artificial Mitochondrial Transfer or Transplant (AMT/T) can be used to reduce the stress and loss of viability of damaged cells. In MitoCeption, a type of AMT/T, the isolated mitochondria and recipient cells are centrifuged together at 4 °C and then co-incubated at 37 °C in normal culture conditions, inducing the transfer. Ultraviolet radiation (UVR) can affect mitochondria and other cell structures, resulting in tissue stress, aging, and immunosuppression. AMT/T could be used to repair UVR cellular and mitochondrial damage. We studied if a mitochondrial mix from different donors (Primary Allogeneic Mitochondrial Mix, PAMM) can repair UVR damage and promote cell survival. Using a simplified adaption of the MitoCeption protocol, we used peripheral blood mononuclear cells (PBMCs) as the recipient cell model of the PAMM in order to determine if this protocol could repair UVR damage. Our results showed that when PBMCs are exposed to UVR, there is a decrease in metabolic activity, mitochondrial mass, and mtDNA sequence stability as well as an increase in p53 expression and the percentage of dead cells. When PAMM MitoCeption was used on UVR-damaged cells, it successfully transferred mitochondria from different donors to distinct PBMCs populations and repaired the observed UVR damage. Our results represent an advancement in the applications of MitoCeption and other AMT/T. We showed that PBMCs could be used as a PAMM source of mitochondria. We also showed that these mitochondria can be transferred in a mix from different donors (PAMM) to UVR-damaged, non-adherent primary cells. Additionally, we decreased the duration of the MitoCeption protocol.

    更新日期:2019-11-18
  • Purification and biochemical characterization of a novel thermostable protease from the oyster mushroom Pleurotus sajor-caju strain CTM10057 with industrial interest
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-07-01
    Maroua Omrane Benmrad; Sondes Mechri; Nadia Zaraî Jaouadi; Mouna Ben Elhoul; Hatem Rekik; Sami Sayadi; Samir Bejar; Nabil Kechaou; Bassem Jaouadi

    Proteases are hydrolytic enzymes that catalyze peptide linkage cleavage reactions at the level of proteins and peptides with different degrees of specificity. This group draws the attention of industry. More than one protease in three is a serine protease. Classically, they are active at neutral to alkaline pH. The serine proteases are researched for industrial uses, especially detergents. They are the most commercially available enzyme group in the world market. Overall, fungi produced extracellular proteases, easily separated from mycelium by filtration. A new basidiomycete fungus CTM10057, a hyperproducer of a novel protease (10,500 U/mL), was identified as Pleurotus sajor-caju (oyster mushroom). The enzyme, called SPPS, was purified to homogeneity by heat-treatment (80 °C for 20 min) followed by ammonium sulfate precipitation (35–55%)-dialysis, then UNO Q-6 FPLC ion-exchange chromatography and finally HPLC-ZORBAX PSM 300 HPSEC gel filtration chromatography, and submitted to biochemical characterization assays. The molecular mass was estimated to be 65 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion by HPLC. A high homology with mushroom proteases was displayed by the first 26 amino-acid residues of the NH2-terminal aminoacid sequence. Phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP) strongly inhibit SPPS, revealing that it is a member of the serine-proteases family. The pH and temperature optima were 9.5 and 70 °C, respectively. Interestingly, SPPS possesses the most elevated hydrolysis level and catalytic efficiency in comparison with SPTC, Flavourzyme® 500 L, and Thermolysin type X proteases. More remarkably, a high tolerance towards organic solvent tolerance was exhibited by SPPS, together with considerable detergent stability compared to the commercial proteases Thermolysin type X and Flavourzyme® 500 L, respectively. This proves the excellent proprieties characterizing SPPS, making it a potential candidate for industrial applications especially detergent formulations.

    更新日期:2019-11-18
  • Modulation of chimeric antigen receptor surface expression by a small molecule switch
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-07-03
    Alexandre Juillerat; Diane Tkach; Brian W. Busser; Sonal Temburni; Julien Valton; Aymeric Duclert; Laurent Poirot; Stéphane Depil; Philippe Duchateau

    Engineered therapeutic cells have attracted a great deal of interest due to their potential applications in treating a wide range of diseases, including cancer and autoimmunity. Chimeric antigen receptor (CAR) T-cells are designed to detect and kill tumor cells that present a specific, predefined antigen. The rapid expansion of targeted antigen beyond CD19, has highlighted new challenges, such as autoactivation and T-cell fratricide, that could impact the capacity to manufacture engineered CAR T-cells. Therefore, the development of strategies to control CAR expression at the surface of T-cells and their functions is under intense investigations. Here, we report the development and evaluation of an off-switch directly embedded within a CAR construct (SWIFF-CAR). The incorporation of a self-cleaving degradation moiety controlled by a protease/protease inhibitor pair allowed the ex vivo tight and reversible control of the CAR surface presentation and the subsequent CAR-induced signaling and cytolytic functions of the engineered T-cells using the cell permeable Asunaprevir (ASN) small molecule. The strategy described in this study could, in principle, be broadly adapted to CAR T-cells development to circumvent some of the possible hurdle of CAR T-cell manufacturing. This system essentially creates a CAR T-cell with an integrated functional rheostat.

    更新日期:2019-11-18
  • Production of alkaline pectinase: a case study investigating the use of tobacco stalk with the newly isolated strain Bacillus tequilensis CAS-MEI-2-33
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-07-12
    Ge Zhang; Shugui Li; Yingbo Xu; Juan Wang; Fan Wang; Yuhua Xin; Zhong Shen; Haibo Zhang; Ming Ma; Haobao Liu

    Tobacco stalk (TS), a major agricultural waste abundant in pectin, has resulted in concerns about the need for its reuse. The nicotine in TS is considered a chemical that is to\xic and hazardous to the environment. In this study, Bacillus tequilensis CAS-MEI-2-33 was isolated from cigar wrappers to produce alkaline pectinase using TS. Subsequently, the medium and fermentation conditions for the production of pectinase by B. tequilensis CAS-MEI-2-33 were optimized. The optimal fermentation period, pH of the initial fermentation medium, concentration of TS, and inoculum amount for B. tequilensis CAS-MEI-2-33 were 40 h, 40 g/L, 7.0, and 3%, respectively. Under optimal conditions, the pectinase activity was 1370 U/mL. Then, the enzymatic properties, such as the optimum pH, reaction temperature, temperature stability, and effects of metal ions, were studied. The optimal pH was determined to be 10.0, indicating that the enzyme was an alkaline pectinase. The optimal temperature was 40 °C, and pectinase activity was stable at 40 °C. The Ag+ metal ions were shown to remarkably promote enzyme activity. The pectinase was partly purified by ammonium sulfate precipitation, ion exchange chromatography, and Sephacryl S-100 chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and LC-MS/MS analyses were utilized to analyze the pectinase. This study provided a new alkaline pectinase candidate and a new strategy for the use of TS.

    更新日期:2019-11-18
  • Enhanced Natamycin production by Streptomyces natalensis in shake-flasks and stirred tank bioreactor under batch and fed-batch conditions
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-07-16
    Elsayed Ahmed Elsayed; Mohamed A. Farid; Hesham A. El-Enshasy

    Natamycin is an antifungal polyene macrolide antibiotic with wide applications in health and food industries. Currently, it is the only antifungal food additive with the GRAS status (Generally Regarded as Safe). Natamycin production was investigated under the effect of different initial glucose concentrations. Maximal antibiotic production (1.58 ± 0.032 g/L) was achieved at 20 g/L glucose. Under glucose limitation, natamycin production was retarded and the produced antibiotic was degraded. Higher glucose concentrations resulted in carbon catabolite repression. Secondly, intermittent feeding of glucose improved natamycin production due to overcoming glucose catabolite regulation, and moreover it was superior to glucose-beef mixture feeding, which overcomes catabolite regulation, but increased cell growth on the expense of natamycin production. Finally, the process was optimized in 7.5 L stirred tank bioreactor under batch and fed-batch conditions. Continuous glucose feeding for 30 h increased volumetric natamycin production by about 1.6- and 1.72-folds in than the batch cultivation in bioreactor and shake-flasks, respectively. Glucose is a crucial substrate that significantly affects the production of natamycin, and its slow feeding is recommended to alleviate the effects of carbon catabolite regulation as well as to prevent product degradation under carbon source limitation. Cultivation in bioreactor under glucose feeding increased maximal volumetric enzyme production by about 72% from the initial starting conditions.

    更新日期:2019-11-18
  • Preparation of a novel monoclonal antibody against caprine interleukin-17A and its applications in immunofluorescence and immunohistochemistry assays
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-07-17
    Yang Gao; Feng Feng Sang; De Lan Meng; Yi Wang; Wen Tao Ma; De Kun Chen

    Interleukin-17 (IL-17), the characteristic cytokine secreted by T helper 17 lymphocytes (Th17 cells), plays a pivotal role in host defense and many inflammatory or autoimmune diseases. The aim of this study was to obtain purified protein caprine IL-17A (cIL-17A) as an antigen for preparing an IL-17A-specific monoclonal antibody (mAb). The coding sequence (CDS) region of cIL-17A was cloned from the peripheral blood mononuclear cells (PBMCs) of dairy goats and then inserted into the expression vector PET 32a and transformed into competent TransB (DE3) cells. Recombinant fusion protein obtained under optimized conditions was used to immunize BALB/c mice for preparing monoclonal antibodies. Finally, the supernatants of two hybridoma cell lines showing positive reaction with the recombinant fusion protein and negative reaction with fusion tags of PET 32a were collected for western blot, immunofluorescence (IF) and immunohistochemistry (IHC) analysis. Our results showed that the maximum amount of soluble protein could be obtained directly in the supernatant when the recombinant expression cells were induced by isopropyl-β-d-thiogalactoside (IPTG) at a concentration of 0.3 mmol/L at 16 °C for 42 h. Western blot analysis showed that the mAb H8 could recognize the eukaryotically expressed cIL-17A in the supernatant of transfected HEK293T cells. Immunofluorescence and immunohistochemistry assays showed that mAb H8 could strongly recognize both the eukaryotically expressed and natural cIL-17A. The monoclonal antibody mAb H8 prepared in this study may be a potential tool for the detection of cIL-17A and beneficial for investigating the pathogenesis of various IL-17-associated diseases.

    更新日期:2019-11-18
  • Crosslinked flagella as a stabilized vaccine adjuvant scaffold
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-07-18
    Casey M. Gries; Rohith R. Mohan; Dimitrios Morikis; David D. Lo

    Engineered vaccine proteins incorporating both antigen and adjuvant components are constructed with the aim of combining functions to induce effective protective immunity. Bacterial flagellin is a strong candidate for an engineered vaccine scaffold as it is known to provide adjuvant activity through its TLR5 and inflammasome activation. Moreover, polymerized flagellin filaments can elicit a more robust immunoglobulin response than monomeric flagellin, and the multimeric antigen form can also promote T cell-independent antibody responses. Here, we aim to produce and test a covalently stabilized polymerized flagellar filament, providing additional immune efficacy through stabilization of its polymeric filament structure, as well as stabilization for long-term storage. Computational modeling of monomer packing in flagellin filaments helped identify amino acids with proximity to neighboring flagella protofilaments. Paired cysteine substitutions were made at amino acids predicted to form inter-monomer disulfide cross-links, and these substitutions were capable of forming flagella when transfected into a flagellin-negative strain of Salmonella enterica subspecies Typhimurium. Interestingly, each paired substitution stabilized different helical conformational polymorphisms; the stabilized filaments lost the ability to transition between conformations, reducing bacterial motility. More importantly, the paired substitutions enabled extensive disulfide cross links and intra-filament multimer formation, and in one of the three variants, permitted filament stability in high acidic and temperature conditions where wild-type filaments would normally rapidly depolymerize. In addition, with regard to potential adjuvant activity, all crosslinked flagella filaments were able to induce wild-type levels of epithelial NF-κB in a cell reporter system. Finally, bacterial virulence was unimpaired in epithelial adherence and invasion, and the cysteine substitutions also appeared to increase bacterial resistance to oxidizing and reducing conditions. We identified amino acid pairs, with cysteine substitutions, were able to form intermolecular disulfide bonds that stabilized the resulting flagellar filaments in detergent, hydrochloric acid, and high temperatures while retaining its immunostimulatory function. Flagellar filaments with disulfide-stabilized protofilaments introduce new possibilities for the application of flagella as a vaccine adjuvant. Specifically, increased stability and heat tolerance permits long-term storage in a range of temperature environments, as well as delivery under a range of clinical conditions.

    更新日期:2019-11-18
  • Quantitative assessment of LASSO probe assembly and long-read multiplexed cloning
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-07-24
    Syukri Shukor; Alfred Tamayo; Lorenzo Tosi; H. Benjamin Larman; Biju Parekkadan

    Long Adapter Single-Stranded Oligonucleotide (LASSO) probes were developed as a novel tool for massively parallel cloning of kilobase-long genomic DNA sequences. LASSO dramatically improves the capture length limit of current DNA padlock probe technology from approximately 150 bps to several kbps. High-throughput LASSO capture involves the parallel assembly of thousands of probes. However, malformed probes are indiscernible from properly formed probes using gel electrophoretic techniques. Therefore, we used next-generation sequencing (NGS) to assess the efficiency of LASSO probe assembly and how it relates to the nature of DNA capture and amplification. Additionally, we introduce a simplified single target LASSO protocol using classic molecular biology techniques for qualitative and quantitative assessment of probe specificity. A LASSO probe library targeting 3164 unique E. coli ORFs was assembled using two different probe assembly reaction conditions with a 40-fold difference in DNA concentration. Unique probe sequences are located within the first 50 bps of the 5′ and 3′ ends, therefore we used paired-end NGS to assess probe library quality. Properly mapped read pairs, representing correctly formed probes, accounted for 10.81 and 0.65% of total reads, corresponding to ~ 80% and ~ 20% coverage of the total probe library for the lower and higher DNA concentration conditions, respectively. Subsequently, we used single-end NGS to correlate probe assembly efficiency and capture quality. Significant enrichment of LASSO targets over non-targets was only observed for captures done using probes assembled with a lower DNA concentration. Additionally, semi-quantitative polyacrylamide gel electrophoresis revealed a ~ 10-fold signal-to-noise ratio of LASSO capture in a simplified system. These results suggest that LASSO probe coverage for target sequences is more predictive of successful capture than probe assembly depth-enrichment. Concomitantly, these results demonstrate that DNA concentration at a critical step in the probe assembly reaction significantly impacts probe formation. Additionally, we show that a simplified LASSO capture protocol coupled to PAGE (polyacrylamide gel electrophoresis) is highly specific and more amenable to small-scale LASSO approaches, such as screening novel probes and templates.

    更新日期:2019-11-18
  • Highly efficient preparation of active S-phenyl-L-cysteine with tryptophan synthase using a chemoenzymatic method
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-07-18
    Lisheng Xu; Xingtao Zhang; Guizhen Gao; Sun Yue

    S-Phenyl-L-cysteine is regarded as having potential applicability as an antiretroviral/protease inhibitor for human immunodeficiency virus (HIV). In the present study, optically active S-phenyl-L-cysteine was prepared in a highly efficient manner from inexpensive bromobenzene using tryptophan synthase through a chemoenzymatic method. The chemoenzymatic method used a four-step reaction sequence. The process started with the reaction of magnesium and bromobenzene, followed by a Grignard reaction, and then hydrolysis and enzymatic synthesis using tryptophan synthase. Through this approach, S-phenyl-L-cysteine was chemoenzymatically synthesized using tryptophan synthase from thiophenol and L-serine as the starting material. High-purity, optically active S-phenyl-L-cysteine was efficiently and inexpensively obtained in a total yield of 81.3% (> 99.9% purity).

    更新日期:2019-11-18
  • Mutagenesis of N-terminal residues confer thermostability on a Penicillium janthinellum MA21601 xylanase
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-07-25
    Ke Xiong; Jie Hou; Yuefeng Jiang; Xiuting Li; Chao Teng; Qin Li; Guangsen Fan; Ran Yang; Chengnan Zhang

    A mesophilic xylanase PjxA from Penicillium janthinellum MA21601 has high specific activity under acidic condition and holds great potential for applications in the animal feed industry. To enhance the thermostability of xylanase PjxA, two mutation strategies in the N-terminal region were examined and then integrated into the xylanase to further improvement. The recombinant xylanase PTxA-DB (The meaning of DB is disulfide-bridge.) was constructed by replacement of five residues in the mutated region in TfxA (T10Y, N11H, N12D, Y15F, N30 L), combined with an additional disulfide bridge in the N-terminal region. The Tm value of mutant PTxA-DB was improved from 21.3 °C to 76.6 °C, and its half-life was found to be 53.6 min at 60 °C, 107-fold higher than the wild type strain. The location of the disulfide bridge (T2C-T29C) was between the irregular loop and the β-strand A2, accounting for most of the improvement in thermostability of PjxA. Further analysis indicated T2C, T29C, N30 L and Y15F lead to increase N-terminal hydrophobicity. Moreover, the specific activity and substrate affinity of PTxA-DB were also enhanced under the acidic pH values. These results indicated PTxA-DB could be a prospective additive to industrial animal feeds.

    更新日期:2019-11-18
  • Enhancement of using combined packing materials on the removal of mixed sulfur compounds in a biotrickling filter and analysis of microbial communities
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-07-25
    Xiang Tu; Meiying Xu; Jianjun Li; Enze Li; Rongfang Feng; Gang Zhao; Shaobin Huang; Jun Guo

    Packing materials is a critical design consideration when employing biological reactor to treat malodorous gases. The acidification of packing bed usually results in a significant drop in the removal efficiency. In the present study, a biotrickling filter (BTF2) packed with plastic balls in the upper layer and with lava rocks in the bottom layer, was proposed to mitigate the acidification. Results showed that using combined packing materials efficiently enhanced the removal performance of BTF2 when compared with BTF1, which was packed with sole lava rocks. Removal efficiencies of more than 92.5% on four sulfur compounds were achieved in BTF2. Average pH value in its bottom packing bed was about 4.86, significantly higher than that in BTF1 (2.85). Sulfate and elemental sulfur were observed to accumulate more in BTF1 than in BTF2. Analysis of principal coordinate analysis proved that structure of microbial communities in BTF2 changed less after the shutdown but more when the initial pH value was set at 5.5. Network analysis of significant co-occurrence patterns based on the correlations between microbial taxa revealed that BTF2 harbored more diverse microorganisms involving in the bio-oxidation of sulfur compounds and had more complex interactions between microbial species. Results confirmed that using combined packing materials effectively improved conditions for the growth of microorganisms. The robustness of reactor against acidification, adverse temperature and gas supply shutdown was greatly enhanced. These provided a theoretical basis for using mixed packing materials to improve removal performance.

    更新日期:2019-11-18
  • Improved the expression level of active transglutaminase by directional increasing copy of mtg gene in Pichia pastoris
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-07-30
    Xiaoping Song; Changsheng Shao; Yugang Guo; Yajie Wang; Jingjing Cai

    The microbial transglutaminase (MTG) is inactive when only the mature sequence is expressed in Pichia pastoris. Although co-expression of MTG and its N-terminal pro-peptide can obtain the active MTG, the enzyme activity was still low. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number recombinants of P. pastoris are achievable only by cloning of gene concatemers, so methods for rapid and reliable multicopy strains are therefore desirable. The coexpression strains harboring different copies mtg were obtained successfully by stepwise increasing Zeocin concentration based on the rDNA sequence of P. pastoris. The genome of coexpression strains with the highest enzyme activity was analyzed by real-time fluorescence quantitative PCR, and three copies of mtg gene (mtg-3c) was calculated according to the standard curve of gap and mtg genes (gap is regarded as the single-copy reference gene). The maximum enzyme activity of mtg-3c was up to 1.41 U/mL after being inducted for 72 h in 1 L flask under optimal culture conditions, and two protein bands were observed at the expected molecular weights (40 kDa and 5 kDa) by Western blot. Furthermore, among the strains detected, compared with mtg-2c, mtg-6c or mtg-8c, mtg-3c is the highest expression level and enzyme activity, implying that mtg-3c is the most suitable for co-expression pro-peptide and MTG. This study provides an effective strategy for improving the expression level of active MTG by directional increasing of mtg copies in P. pastoris.

    更新日期:2019-11-18
  • Provision of carbon skeleton for lipid synthesis from the breakdown of intracellular protein and soluble sugar in Phaeodactylum tricornutum under high CO2
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-07-26
    Aiyou Huang; Songcui Wu; Wenhui Gu; Yuanxiang Li; Xiujun Xie; Guangce Wang

    Increasing CO2 emissions have resulted in ocean acidification, affecting marine plant photosynthesis and changing the nutrient composition of marine ecosystems. The physiological and biochemical processes of marine phytoplankton in response to ocean acidification have been reported, but have been mainly focused on growth and photosynthetic physiology. To acquire a thorough knowledge of the molecular regulation mechanisms, model species with clear genetic background should be selected for systematic study. Phaeodactylum tricornutum is a pennate diatom with the characteristics of small genome size, short generation cycle, and easy to transform. Furthermore, the genome of P. tricornutum has been completely sequenced. In this study, P. tricornutum was cultured at high and normal CO2 concentrations. Cell composition changes during culture time were investigated. The 13C isotope tracing technique was used to determine fractional labeling enrichments for the main cellular components. The results suggested that when lipid content increased significantly under high CO2 conditions, total protein and soluble sugar contents decreased. The 13C labeling experiment indicated that the C skeleton needed for fatty acid C chain elongation in lipid synthesis under high CO2 conditions is not mainly derived from NaHCO3 (carbon fixed by photosynthesis). This study indicated that breakdown of intracellular protein and soluble sugar provide C skeleton for lipid synthesis under high CO2 concentration.

    更新日期:2019-11-18
  • Isolating promoters from Corynebacterium ammoniagenes ATCC 6871 and application in CoA synthesis
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-11-12
    Yingshuo Hou; Siyu Chen; Jianjun Wang; Guizhen Liu; Sheng Wu; Yong Tao

    Corynebacterium ammoniagenes is an important industrial organism that is widely used to produce nucleotides and the potential for industrial production of coenzyme A by C. ammoniagenes ATCC 6871 has been shown. However, the yield of coenzyme A needs to be improved, and the available constitutive promoters are rather limited in this strain. In this study, 20 putative DNA promoters derived from genes with high transcription levels and 6 promoters from molecular chaperone genes were identified. To evaluate the activity of each promoter, red fluorescence protein (RFP) was used as a reporter. We successfully isolated a range of promoters with different activity levels, and among these a fragment derived from the upstream sequence of the 50S ribosomal protein L21 (Prpl21) exhibited the strongest activity among the 26 identified promoters. Furthermore, type III pantothenate kinase from Pseudomonas putida (PpcoaA) was overexpressed in C. ammoniagenes under the control of Prpl21, CoA yield increased approximately 4.4 times. This study provides a paradigm for rational isolation of promoters with different activities and their application in metabolic engineering. These promoters will enrich the available promoter toolkit for C. ammoniagenes and should be valuable in current platforms for metabolic engineering and synthetic biology for the optimization of pathways to extend the product spectrum or improve the productivity in C. ammoniagenes ATCC 6871 for industrial applications.

    更新日期:2019-11-13
  • Correction to: A new primer construction technique that effectively increases amplification of rare mutant templates in samples
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-11-12
    Jr-Kai Huang; Ling Fan; Tao-Yeuan Wang; Pao-Shu Wu

    Following publication of the original article [1], the author informed us that the legend for Fig. 2 was incorrect.

    更新日期:2019-11-13
  • Rapid generation and selection of Cas9-engineering TRP53 R172P mice that do not have off-target effects
    BMC Biotechnol. (IF 2.303) Pub Date : 2019-11-08
    Guoxing Zheng; Qingqing Zhu; Junchao Dong; Xin Lin; Chengming Zhu

    Genetic mutations cause severe human diseases, and suitable animal models to study the regulatory mechanisms involved are required. The CRISPR/Cas9 system is a powerful, highly efficient and easily manipulated tool for genetic modifications. However, utilization of CRISPR/Cas9 to introduce point mutations and the exclusion of off-target effects in mice remain challenging. TP53-R175 is one of the most frequently mutated sites in human cancers, and it plays crucial roles in human diseases, including cancers and diabetes. Here, we generated TRP53-R172P mutant mice (C57BL/6 J, corresponding to TP53-R175P in humans) using a single microinjection of the CRISPR/Cas9 system. The optimal parameters comprised gRNA selection, donor designation (silent mutations within gRNA region), the concentration of CRISPR components and the cellular sites of injection. TRP53-R172P conversion was genetically and functionally confirmed. Combination of TA cloning and Sanger sequencing helped identify the correctly targeted mice as well as the off-target effects in the engineered mice, which provide us a strategy to select the on-target mice without off-target effects quickly and efficiently. A single injection of the this optimized CRISPR/Cas9 system can be applied to introduce particular mutations in the genome of mice without off-target effects to model various human diseases.

    更新日期:2019-11-11
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