A novel gene delivery system in plants with calcium alginate micro-beads

We have produced micrometer-sized calcium alginate beads referred to as "bio-beads" that encapsulate plasmid DNA molecules carrying a reporter gene. In order to evaluate the efficiency of the bio-beads in mediating genetic transfection, protoplasts isolated from cultured tobacco cells (BY-2) were transfected with bio-beads containing a plasmid that carries the modified green fluorescent protein gene CaMV35S-sGFP. With the bio-beads treatment, approximately ten-fold higher GFP expression was observed after 24 h incubation compared to that with the conventional method using a naked plasmid solution. Transfection was up to 0.22% efficient. These results indicate that bio-beads have a possibility for efficient transformation in plants.     

A microscopic method to visualize Escherichia coli interaction with beef muscle

The genetic determinant for enhanced green fluorescent protein (EGFP) was introduced into Escherichia coli JM109 (ATCC 53323) and E. coli O157:H7 (ATCC 43895) on plasmid EGFP. The expression of EGFP did not change the growth kinetics or surface properties tested (hydrophobicity and electrophoretic mobility). Microscope slides were modified to allow for optimal viewing of thick meat samples with an inverted microscope. Two fluorescent dyes, nile red and Cy3 were used to stain for lipid and protein portions of beef muscle, respectively. Laser scanning confocal microscopy was used to observe interaction of the EGFP-expressing E. coli strains and the fluorescently stained muscle components without changing the spatial and temporal environment of the organisms.     

Incorporation of lipid nanoparticles into calcium alginate beads and characterization of the encapsulated particles by differential scanning calorimetry

The aim of this study was to encapsulate lipid nanoparticle dispersions into calcium alginate microbeads and to investigate the feasibility of this incorporation as well as the stability of the enclosed nanoparticles. Two methods were used to prepare the lipid-containing microbeads by external gelation, i.e. an electrostatic droplet generation technique and a spraying method using the two-fluid spray nozzle of a spray dryer. The particle sizes of the hydrogel beads were determined with laser diffraction. The first method produced particles with median sizes between 330 and 1350 μm, depending on the needle applied. Using the second method smaller particles (∼45 μm) could be prepared. Small tyloxapol-stabilized trimyristin nanoparticles (<100 nm) could easily be incorporated into the differently sized alginate beads. The small size of the lipid nanoparticles leads to a typical melting behavior characterized by the occurrence of multiple discrete melting peaks in their differential scanning calorimetry (DSC) thermograms which enabled the characterization of the lipid particles within the microbeads. Thus, any negative influence of the preparation procedure on the lipid dispersions could be detected and it was possible to verify optimization options of the incorporation procedure. Finally, handling and shelf life of the hydrogel beads were improved by a freezing process.     

Cytotoxicity evaluation of reactive metabolites using rat liver homogenate microsome-encapsulated alginate gel microbeads

We present an improved cytotoxicity test for reactive metabolites, in which the S9 microsomal fraction of rat liver homogenate is encapsulated in alginate gel microbeads to avoid cytotoxic effects of S9-self-generated toxicants, microsomal lipid peroxides. The S9-encapsulated gel microbeads were prepared by a coaxial two-fluid nozzle and surfaces of the microbeads were coated with poly-L-lysine (PLL). Although the initial metabolic rate of the S9-encapsulated gel microbeads was about 20% slower than that of bare S9, the microbeads prevented the leakage of microsomal lipid peroxides thanks to the dense alginate and PLL polymer networks. In fact, the half maximal effective concentration of the indirect mutagen cyclophosphamide on NIH3T3 cells in the presence of the S9-encapsulated gel microbeads was about 5 times higher than that in the presence of bare S9. Use of the S9-encapsulated gel microbeads enabled the more accurate evaluation of the cytotoxicity of the reactive metabolites without the S9-based cytotoxicity.     

Expression of red-shifted green fluorescent protein by Escherichia coli O157:H7 as a marker for the detection of cells on fresh produce

Escherichia coli O157:H7 was transformed with a plasmid vector red-shifted green fluorescence protein (pEGFP) to express red-shifted green fluorescence protein (EGFP) from Aequorea victoria. The EGFP expression among total cells and nonviable cells was determined at the cellular level by microscopic observation of immunostained and membrane-impermeable, dye-stained cultures, respectively. E. coli O157:H7 retained pEGFP during frozen storage at -80 degrees C. The percentage of EGFP expression was improved by repeated subculturing, reaching 83.4 +/- 0.1%, although the fluorescence intensity varied among cells. A low percentage of EGFP-expressing cells was nonviable. The percentage of EGFP decreased when the culture plate was kept at 4 degrees C, suggesting that some cells lost pEGFP during refrigeration. The storage of the culture suspension in sterile deionized water at 4 degrees C for 24 h reduced the percentage of EGFP expression, indicating that some EGFP was denatured. The application of EGFP as a marker for E. coli O157:H7 on green leaf lettuce, cauliflower, and tomato was evaluated using confocal scanning laser microscopy. EGFP-transformed cells were readily visible under confocal scanning laser microscopy on all produce types. The numbers of E. coli O157:H7 cells detected with EGFP were equivalent to those detected with immunostaining for green leaf lettuce and cauliflower but less for tomato. E. coli O157:H7 attached preferentially to damaged tissues of green leaf lettuce and tomato over intact tissue surfaces and to flowerets of cauliflower than to stem surfaces. EGFP can serve as a marker to characterize E. coli O157:H7 attachment on green leaf lettuce and cauliflower but may not be suitable on tomato.     

Calcium–Alginate–Inulin Microbeads as Carriers for Aqueous Carqueja Extract

Carqueja (Pterospartum tridentatum) is an endemic species and various bioactive compounds have been identified in its aqueous extract. The aim of this study was to protect the natural antioxidants from the aqueous extract of carqueja by encapsulation in Ca–alginate microbeads and Ca–alginate microbeads containing 10% and 20% (w/v) of inulin. The microbeads produced by electrostatic extrusion technique had an average diameter from 625 μm to 830 μm depending on the portion of inulin. The sphericity factor of the hydrogel microbeads had values between 0.014 and 0.026, while freeze dried microbeads had irregular shape, especially those with no excipient. The reduction in microbeads size after freeze drying process (expressed as shrinkage factor) ranged from 0.338 (alginate microbeads with 20% (w/v) of inulin) to 0.523 (plain alginate microbeads). The expressed radical scavenging activity against ABTS and DPPH radicals was found to be between 30% and 40% for encapsulated extract, while the fresh extract showed around 47% and 57% of radical scavenging activity for ABTS and DPPH radicals, respectively. The correlation between antioxidant activity and the total phenolic content were found to be positive (in both assay methods, DPPH and ABTS), which indicate that the addition of inulin didn't have influence on antioxidant activity. The presence of inulin reduced stiffness of the hydrogel, and protected bead structure from collapse upon freeze‐drying. Alginate–inulin beads are envisaged to be used for delivery of aqueous P. tridentatum extract in functional food products.     

Method for reverse transfection using gold colloid as a nano-scaffold

DNA microarray of non-viral reverse transfection in cell engineering allows drastic downsizing of large-scale functional screening of genes and siRNAs. However the control of localizability and efficiency of the microarray is still considered as a critical barrier in practical use. One of the major breakthrough to increase the transfection efficiency may be control in the condition of DNA/transfection reagent complex on the microarray surface. In this paper, we showed that negatively charged gold colloid (GC) is successfully used to control the DNA/reagent complex on a glass surface. The conjugation of gold nanoparticles (20 nm in diameter) to the pEGFP-N1/Jet-PEI complex resulted in a more than 2.5-fold increase in the intensity of fluorescence of enhanced green fluorescent protein (EGFP) (based on the efficiency of transfection) from human mesenchymal stem cells (hMSCs), as compared to the control without GC. Our method for reverse transfection should be useful not only for cell array-based analyses but also as a novel gene-delivery method for gene therapy in regenerative medicine.     

Polyethyleneimine/chitosan hexamer-mediated gene transfection into intestinal epithelial cell cultured in serum-containing medium

In search of an efficient nonviral vector, polyethyleneimine (PEI)-based vectors were examined. In general, the transfection efficiency of nonviral vectors is suppressed by serum. Here we show that PEI based vectors, particularly, the chitosan hexamer-PEI vector, could perform efficient gene transfection into intestinal epithelial cells (IEC-6) in the presence of serum. The conjugation order of the two polymers with a plasmid (first, chitosan hexamer; second, PEI) was found to be an important factor in enhancing transfection efficiency.     

Selection of highly productive mammalian cells based on an inducible growth advantage using an antibody/receptor chimera

In mammalian cell culture, the selection of high producers is a critical step in efficient recombinant protein production. Drug-resistance selection has been commonly used, but does not always give a pure population of high producers. In this study, we propose a novel selection method in which the growth of high producers is specifically promoted. Two plasmids encoding (i) a hybrid receptor composed of the V(H) portion of anti-hen egg lysozyme antibody HyHEL-10 and an N-terminally truncated erythropoietin receptor (V(H)-EpoR), and (ii) a V(L)-EpoR fusion derived from the same construct as in (i), were employed. The second plasmid contained enhanced green fluorescent protein (EGFP) as a model recombinant protein that was flanked by the internal ribosomal entry sequence. Both plasmids were used simultaneously to transfect an IL-3-dependent murine myeloid cell line, 32D. The transfectants, after antigen selection in the absence of IL-3, showed a clear antigen-induced dose-dependent proliferation. In addition, a high EGFP expression level was observed by flow cytometry in comparison with the cells before antigen selection. The results clearly demonstrate the advantage of our method over conventional drug-resistance selection. We propose the term AMEGA (Antigen MEdiated Genetically-modified cell Amplification) for such an approach.     

A conditional suicide system for Saccharomyces cerevisiae relying on the intracellular production of the Serratia marcescens nuclease

A conditional lethal system for biological containment of genetically modified strains of Saccharomyces cerevisiae is described. This suicide system is based on the intracellular production of the Serratia marcescens nuclease in the yeast cell, aiming at the destruction of the host genetic material. The S. marcescens nuclease, encoded by the nucA gene, is normally secreted by the bacterium into the medium. In the present work, the nucA gene, devoid of its signal peptide coding sequence, was cloned in a yeast expression vector, under control of the glucose-repressed S. cerevisiae alcohol dehydrogenase 2 gene (ADH2) promoter. When transformed into S. cerevisiae, the recombinant plasmid proved to be effective in killing the host cells upon glucose depletion from the medium, and the nuclease activity was found in lysates prepared from the transformants. In addition, the nuclease degrading effect was shown to reach chromosomal DNA in the yeast host. The killing effect of the nucA plasmid was also demonstrated in soil microcosm assays, indicating that whenever the GMM escapes into the environment where glucose is scarce, the nucA gene will be expressed and the resulting nuclease will destroy the genetic material and kill the cells. In contrast to other suicide systems that target the cell envelope, the advantage of the one described here is that it disfavours horizontal gene transfer from recombinant yeast cells to other microorganisms found in the environment.     

YHp as a highly stable,hyper-copy,hyper-expression plasmid constructed using a full 2-μm circle sequence in cir0 strains of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the yeast episomal plasmid (YEp), containing a partial sequence from a natural 2-μm plasmid, has been frequently used to induce high levels of gene expression. In this study, we used Japanese sake yeast natural cir⁰ strain as a host for constructing an entire 2-μm plasmid with an expression construct using the three-fragment gap-repair method without Escherichia coli manipulation. The 2-μm plasmid contains two long inverted repeats, which is problematic for the amplification by polymerase chain reaction. Therefore, we amplified it by dividing into two fragments, each containing a single repeat together with an overlapping sequence for homologous recombination. TDH3 promoter-driven yEmRFP (TDH3p-yEmRFP) and the URA3 were used as a reporter gene and a selection marker, respectively, and inserted at the 3′ end of the RAF1 gene on the 2-μm plasmid. The three fragments were combined and used for the transformation of sake yeast cir⁰ ura3^- strain. The resulting transformant colonies showed a red or purple coloration, which was significantly stronger than that of the cells transformed with YEp-TDH3p-yEmRFP. The 2-μm transformants were cultured in YPD medium and observed by fluorescence microscopy. Almost all cells showed strong fluorescence, suggesting that the plasmid was preserved during nonselective culture conditions. The constructed plasmid maintained a high copy state similar to that of the natural 2-μm plasmid, and the red fluorescent protein expression was 54 fold compared with the chromosomal integrant. This vector is named YHp, the Yeast Hyper expression plasmid.     

Possible gene interchange between plasmid and chromosome in yeast

Genomic DNAs isolated from 420 yeast strains stocked in the Department of Fermentation Technology, Hiroshima University (HUT) were screened for the presence of a plasmid sequence both as plasmid or in the chromosome. Five DNA samples gave rise to a positive hybridization signal when 32P-labelled Zygosaccharomyces plasmid pSR1 was used as a probe. Two among these contain hybridizing sequences as plasmids while the other three apparently were chromosomal. Two chromosomal DNA segments of HUT 7195 (Zygosaccharomyces spp.) which hybridized with pSR1 probe were cloned and sequenced. Both DNAs hybridized with a plasmid sequence covering the P gene of pSR1. One of the two segments contains a large open reading frame which can encode 410 amino acid residues. The deduced amino acid sequence is closely related with that of the P gene of pSR1. The present finding suggests that there was an interchange(s) of a gene between yeast plasmid(s) and chromosomes.     

Chemometric evaluation of binary mixtures of alginate and polysaccharide biopolymers as carriers for microencapsulation of green tea polyphenols

In this study principal component analysis and artificial neural networks were used to evaluate the potential of using binary mixtures of sodium alginate and other polysaccharide biopolymers as the carriers for microencapsulation of green tea bioactive compounds. Using binary mixtures of alginate and adjunct biopolymers increased the particle size (from 722 to 1344 µm) and textural parameters of the microbeads. Chemometric techniques revealed the combination of biopolymers and their ratio as the main factors influencing the encapsulation performance. The combination of alginate with hydroxypropyl methylcellulose and locust bean gum enabled to retain the highest (-)-epigallocatechin gallate and caffeine contents, the highest total phenols encapsulation efficiency, and their most retarded release in water, confirming these as the best delivery systems of polyphenol-type active compounds and signifying their potent food applications.     

Effect of poly(beta-hydroxybutyrate) accumulation on the stability of a recombinant plasmid in Escherichia coli

Poly(beta-hydroxybutyrate) (PHB) production using a recombinant Escherichia coli VG1 (pTU14) was carried out. PHB granules were gradually biosynthesized in recombinant cells as inclusion bodies and the morphology of cells became linearly inflated with PHB accumulation from 6 h to 36 h of culture. The stability of plasmid pTU14 was inevitably affected by in-cell PHB accumulation. During repeated subcultures under both PHB-accumulating and non-PHB-accumulating conditions, plasmid DNAs of pTU14 were extracted and analyzed after more than 60 generations. The results showed that plasmid pTU14 is structurally stable but segregationally unstable, and that the segregational instability worsened with increasing accumulation of PHB granules. This is due to not only the serious metabolic burden of plasmid replication and gene expression on the host cell, but also the volume effects of PHB granules on the natural random distribution of plasmids during the binary fission of cells.     

Characterization of Fish Myofibrillar Protein by Conjugation with Alginate Oligosaccharide Prepared Using Genetic Recombinant Alginate Lyase

ABSTRACT: The production of alginate lyase using genetically modified Escherichia coli was superior to the purification of alginate lyase from a culture medium of Pseudoalteromonas elyakovii regarding production efficiency. When alginate oligosaccharide (AO) prepared using genetic recombinant alginate lyase was introduced to fish myofibrillar proteins, the protein obtained high water solubility and improved thermal stability, similarly to AO prepared using wild-type lyase. Therefore, the use of genetic recombinant technology for the production of alginate lyase would be useful for the functional improvement of fish myofibrillar proteins by conjugation with AO.     

酵母细胞的高效转化方法

运用醋酸锂处理酵母细胞，建立并优化了酵母完整细胞的高效质粒转化体系，转化率达１０４μｇ－１．通过对影响转化的诸因子的研究，发现载运ＤＮＡ是影响酵母完整细胞转化效率的最主要因素，热休克处理及处理时间、聚乙二醇相对分子质量及浓度等对转化率也有显著影响。     

Encapsulation of polyphenolic antioxidants from medicinal plant extracts in alginate–chitosan system enhanced with ascorbic acid by electrostatic extrusion

In this study the encapsulation of raspberry leaf, hawthorn, ground ivy, yarrow, nettle and olive leaf extracts was performed by electrostatic extrusion in alginate-chitosan microbeads, with ascorbic acid being used for the dissolution of chitosan. The original and encapsulated plant extracts were characterized for their polyphenol content and composition, mineral content and antioxidant capacity. Raspberry leaf encapsulating microbeads exhibited the highest total phenol content and antioxidant capacity, followed by hawthorn, while olive leaf microbeads contained the lowest total phenol content. High encapsulation efficiency was obtained for all extract encapsulating microbeads (80-89%). Nettle extract-containing microparticles were characterized with the largest particle size and irregular shape, due to a high content of microelements (copper, strontium, and zinc), which affected the geling process of alginate. Although the antioxidant stability of hydrogel microcapsules was deteriorated during refrigerated storage, which might be attributed to the instability of ascorbic acid, the obtained microbeads deliver significant biological activity and antioxidant potential which may increase the daily intake of antioxidants when implemented in a food product.     

高效表达淀粉酶的工业酿酒酵母菌株的构建

将PCR扩增的扣囊腹膜孢酵母菌淀粉酶基因 ,与磷酸甘油酸激酶基因 1启动子和α 分泌序列一起插入含 2 μm的酵母穿梭质粒YEp3 5 2 ,构建酵母菌重组表达质粒并命名为pLA8α。用醋酸锂转化法转化工业酿酒酵母Sc 1 6,转化子培养上清液的淀粉酶活性为 6 8U/mL ,46%的淀粉被降解 ,SDS PAGE检测到约 5 5ku的蛋白条带。转化子在非选择条件下的遗传稳定性为82 %。