Studies of poly-<Emphasis Type="Italic">L</Emphasis>-lysine-starch nanoparticle preparation and its application as gene carrier

Anion starch nanoparticle (StNP) with a diameter of 50 nm was prepared in water-in-oil microemulsion, with soluble starch as raw materials and POCl₃ as crosslinking agent. PLL-StNP was prepared by linking poly-L-lysine (PLL) on the surface of StNP. At the same time, the size of PLL-StNP and its stability in aqueous solution were checked by AFM. The analysis of plasmid DNA binding, DNase I enzymatic degradation, toxicity and transfection were done. We discovered that PLL-StNP may be used as non-virus nanoparticle gene carrier. And we developed the method of preparing PLL-StNP gene carrier and used it in cell transfection. As non-virus gene carrier, PLL-StNP has some advantages, such as large load of DNA, high transfection efficiency, low cell toxicity and biodegradability.     

Development of a systems analysis approach for resolving the structure of biodegrading soil systems

An experimental and mathematical method is developed for the microbial systems analysis of polyaromatic hydrocarbon (PAH)-degrading mixed cultures in PAH-contaminated “town gas” soil systems. Frequency response is the primary experimental and data analysis tool used to probe the structure of these complicated systems. The objective is to provide a fundamental protocol for evaluating the performance of specific mixed microbial cultures on specific soil systems by elucidating the salient system variables and their interactions. Two well-described reactor systems, a constant volume stirred tank reactor (CSTR) and a plug flow differential volume reactor, are used in order to remove performance effects that are related to reactor type as opposed to system structure. These two reactor systems are well-defined systems that can be described mathematically and represent the two extremes of one potentially important system variable, macroscopic mass transfer. The experimental and mathematical structure of the protocol is described, experimental data is presented, and data analysis is demonstrated for the stripping, sorption, and biodegradation of napththalene.     

CLONING, EXPRESSION AND CHARACTERIZATION OF Pro3 GENE OF YEAST Saccharomyces cerevisiae

The proline biosynthetic pathway and Pro genes in Saccharomyces cerevisiae have just begun to be studied recently. In our laboratory, Pro2 gene of S. cerevisiae had been cloned in yeast. As described in this paper, yeast Pro3 gene was also cloned, which can complement yeast Pro3 mutants, and be expressed efficiently in E. coli. The high activities of this gene product, L-pyrroline-5-carboxylate (P5C) reductase, can be detected in both organisms. The activity of the Pro3 gene product in multiple copy plasmids is not higher than that of single copy genes in chromosomes in both yeast and E. coll. The preliminary characterization of the gene is also reported.     

Novel amphiphilic fluorescent graft copolymers: synthesis,characterization, and potential as gene carriers

Amphiphilic fluorescent graft copolymer (PVP‐PyATAm) was successfully synthesized by the free radical copolymerizations of hydrophobic monomer N‐acryloyl‐thioureylene‐4‐(1‐pyrene)‐butyryl amide (PyATAm) with hydrophilic precursor polymers of vinyl‐functionalized poly (N‐vinylpyrrolidone) (Acryloyl‐PVP) in DMF. FT‐IR, ¹H NMR, TEM, gel permeation chromatography‐multi‐angle laser light scattering, UV‐vis spectroscopy, viscometric measurement, and fluorescence spectroscopy were used to characterize this copolymer. The TEM observation showed that the copolymer PVP‐ PyATAm formed spherical micelles in an aqueous solution and the size of micelles was between 50 and 70 nm in diameter. The interaction of PVP‐PyATAm copolymer and plasmid DNA was examined by agarose gel electrophoresis and TEM. Results indicated that the copolymer–DNA complexes were self‐assembled and the size of complexes was between 90 and 120 nm in diameter. Cytotoxity studies using MTT colorimetric assays suggested good biocompatibility of PVP‐PyATAm in vitro. These results suggested the potential of this graft copolymer as gene delivery carrier. Copyright © 2007 John Wiley & Sons, Ltd.     

Construction of recombinant Bacillus subtilis for production of polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1, and pBE2C1AB, containing one or two PHA synthse, genes, respectively. The two plasmids were inserted into Bacillus subtilis DB104 to generate modified strains, B. subtilis/pBE2C1 and B. subtilis/pBE2C1AB. The two recombinants strains were subjected to fermentation and showed PHA accumulation, the first reported example of mcl-PHA production in B. subtilis. Gas Chromatography analysis identified the compound produced by B. subtilis/pBE2C1 to be a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer whereas that produced by B. subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxyde-canoate-co-hydroxydodecanoate (HB-HD-HDD) polymer.     

Capacity of Bacillus thuringiensis S-layer protein displaying polyhistidine peptides on the cell surface

S-layer protein of Bacillus thuringiensis strain CTC was used as the carrier protein to display polyhistidine (poly[6His]) peptides on the cell surface. Poly(6His) _n was fused with S-layer protein at two different sites, inserting just downstream of the S-layer protein homologous domain (slh) and replacing the non-slh region of S-layer protein, respectively. The two series chimeric proteins were both expressed by crystal negative B. thuringiensis strain 4Q7 and strain 171, respectively, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recombinant B. thuringiensis cells gained Ni²⁺- and Cd²⁺-binding ability and had a capacity to display up to nine copies of poly(6His). The Cd²⁺ adsorption quantity of the recombinant strain with the strongest adsorption ability was twice that of the host strain.     

Cyclodextrin-Containing Polymers for Gene Delivery

Cyclodextrin-containing polymers are now being explored as vehicles for delivering nucleic acids into cells. The structures of the cyclodextrin-containing polycations affect the nucleic acid delivery efficiencies and their toxicities. Of interest is the fact that the cyclodextrin-containing polymers reveal lower toxicities than polymers that lack the cyclodextrins. The cyclodextrins endow the nucleic acid delivery vehicles with the ability to be modified by compounds that form inclusion complexes with the cyclodextrins, and these modifications can be performed without disruption of the polymer-nucleic acid interactions. Thus, cyclodextrin-containing polymers provide unique properties for gene delivery.     

毛细管电泳-单链构象多态性分析检测K-ras基因突变

K ras癌基因的点突变在结直肠癌的发生起重要作用。以异丙醇为聚合反应链转移剂,水相法合成 特性粘度为0.70×10-3m3/kg,分子量为6.5×104的低粘度短链线性聚丙烯酰胺。以6%线性聚丙烯酰胺为 筛分介质,分离温度27℃,分离电压9kV为电泳条件,建立了检测K ras基因突变的毛细管电泳 单链构象多 态性方法。利用该方法检测36例结直肠癌患者肿瘤组织,发现12例K ras基因突变。结果表明:该方法具有 快速、高灵敏的优点,为大规模进行结直肠肿瘤的早期诊断提供了可靠方法。