Effect of the kanamycin resistance marker on stability of 2μ-based expression plasmids

U ovom radu opisan je uticaj gena za kanamicinsku rezistenciju (Kmr) na održavanje 2μm plazmida u Saccharomyces cerevisiae. Prisustvo ovog marker gena dovodi do gubitka stabilnog model-vektora konstantnom stopom nezavisnom od izvora ugljenika i stope rasta kulture. Kod sojeva za sintezu heterolognih proteina sa galaktoznog promotora (GALUAS) uvođenje Kmr rezultira "čišćenjem" od kvaščevih epizomalnih plazmida (YEp) u svega nekoliko generacija. Primena selektivnog pritiska na sojeve koji proizvode rekombinantnu penicilin G amidazu (rPGA) nije dovela do očekivanog povećanja prinosa proteina. Ispitivanjem uticaja samih genetičkih elemenata za proizvodnju heterolognih proteina na stabilnost vektora pokazano je da najjače destabilišuće dejstvo ima prisustvo i eksprecija stranog gena.In this paper we describe the effect of the kanamycin resistance gene (Kmr) on 2μm-based plasmid maintenance in Saccharomyces cerevisiae. The influence of this marker gene on the loss of the stable model-vectors proved to be constant, as well as independent of carbon source and culture growth rates. In strains for GALUAS - driven heterologous protein production introduction of Kmr resulted in curing of the yeast episomal plasmid (YEp) from the population in a small number of generations. Application of selective pressure on the strain producing recombinant penicillin G amidase (rPGA) did not provide the expected increase of protein yield. The influence of genetic elements for heterologous protein production on vector stability was examined, and the most destabilizing factors prove to be the presence and expression of the foreign gene

Isolation and characterization of bioactive secondary metabolites from selected Streptomyces strains

Actinomycetes are Gram-positive soil bacteria known as producers of secondary bioactive metabolites. In the search for new bioactive compounds collection of actinomycetes from the Laboratory for Microbial Molecular Genetics and Ecology (Institute of Molecular Genetic and Genetic Engineering, University of Belgrade) was screened according to several criteria - bioactivity, the presence of the genes for polyketide synthase (PKS), pigmentation, and crude culture extracts absorption profile in ultraviolet and visible (UV/Vis) spectra. Based on these we have isolated three strains NP10, JS520, and Streptomyces durmitotensis, previously renewed for didehydroroflamicoin (DDHR) production. Isolates NP10 and JS520 were identified to belong to the genus Streptomyces. Streptomyces sp. NP10 accumulated branched chain fatty acids, predominately isopalmitic, and cyclic dipeptides consisting of proline in combination with valine, isoleucine or alanine, that have a stimulating effect on the trophoblast cell line in culture at low concentrations (1 ng/ml and 1 mg/ml) and cytotoxic effect at high concentrations (1 mg/ml). Streptomyces sp. JS520 was determined to be exceptionally good producer of pigment undecylprodigiosine (UP) with antibacterial, antifungal, antioxidant and UV protective activities showing cytotoxic effects on trophoblast cell line in culture at high concentrations (1 ng/ml). Cytotoxicity of polyene macrolide antibiotic DDHR from S. durmitotensis was confirmed and it’s antifungal effects were shown. PKS cluster responsible for DDHR synthesis was subcloned in the cosmid library and partially sequenced. Optimization of culture conditions resulted in 2.12 times increased production of UP, and 1.1 times increased production of DDHR.Actinomycetes are Gram-positive soil bacteria known as producers of secondary bioactive metabolites. In the search for new bioactive compounds collection of actinomycetes from the Laboratory for Microbial Molecular Genetics and Ecology (Institute of Molecular Genetic and Genetic Engineering, University of Belgrade) was screened according to several criteria - bioactivity, the presence of the genes for polyketide synthase (PKS), pigmentation, and crude culture extracts absorption profile in ultraviolet and visible (UV/Vis) spectra. Based on these we have isolated three strains NP10, JS520, and Streptomyces durmitotensis, previously renewed for didehydroroflamicoin (DDHR) production. Isolates NP10 and JS520 were identified to belong to the genus Streptomyces. Streptomyces sp. NP10 accumulated branched chain fatty acids, predominately isopalmitic, and cyclic dipeptides consisting of proline in combination with valine, isoleucine or alanine, that have a stimulating effect on the trophoblast cell line in culture at low concentrations (1 ng/ml and 1 mg/ml) and cytotoxic effect at high concentrations (1 mg/ml). Streptomyces sp. JS520 was determined to be exceptionally good producer of pigment undecylprodigiosine (UP) with antibacterial, antifungal, antioxidant and UV protective activities showing cytotoxic effects on trophoblast cell line in culture at high concentrations (1 ng/ml). Cytotoxicity of polyene macrolide antibiotic DDHR from S. durmitotensis was confirmed and it’s antifungal effects were shown. PKS cluster responsible for DDHR synthesis was subcloned in the cosmid library and partially sequenced. Optimization of culture conditions resulted in 2.12 times increased production of UP, and 1.1 times increased production of DDHR

Glycosylation and pH stability of penicillin G acylase from providencia rettgeri produced in Pichia pastoris

Penicilin G acilaza (PAC) je jedan od najšire korišćenih enzima u industrijskoj sintezi polusintetskih antibiotika. U ovom radu dobijeni nivo ekspresije PAC gena iz Providencia rettgeri u ekspresionom sistemu Pichia pastoris iznosio je 2.7 U/ml. Rekombinantni enzim je prečišćen i određen je njegov glikozilacioni status. Nađeno je da osim što su obe subjedinice enzima (α i β) N-glikozilovane, β subjedinica sadrži još i O-glikane. Takođe je ustanovljeno da je rekombinantna PACP. rett. stabilna u širokom pH opsegu što ju je, zajedno sa predhodno ustanovljenom visokom termostabilnošću, učinilo izuzetno privlačnim biokatalizatorom sa industrijske tačke gledišta.Penicillin G acylase (PAC) is one of the most widely used enzymes in industrial synthesis of semi-synthetic antibiotics. The Providencia rettgeri pac gene was expressed to a level of 2.7 U/ml using the Pichia pastoris expression system. The recombinant enzyme was purified and its glycosylation status was determined. It was found that both subunits (α and β) of the enzyme were N-glycosylated, while the β-subunit also contained O-glycans. It was also observed that rPACP.rett. was stable in a wide range of pH, which, in addition to the previously proved high thermostability, makes it an attractive biocatalyst from an industrial point of view

Potential of environmental Serratia from herbicide treated soil for pathogenicity in humans

Soils contain microorganisms capable of causing diseases in humans, either as opportunistic or as obligate pathogens. The application of herbicides increases the prevalence of antibiotic resistance genes and mobile genetic elements, which expands the competitive niche of pathogenic microbes. This implies that herbicides could impose selective pressure driving the spread of antibiotic resistance and therefore making heavily herbicide treated agricultural soil a possible reservoir of multiresistant opportunistic human pathogens. From the soil exposed to chlorinated aromatic herbicides S-metolachlor and isoxaflutole, semi-persistent selective herbicides widely used in European Union and Serbia we isolated bacteria able to grow on herbicide as the sole carbon source. Bacterial isolates were assessed for various virulence traits and resistance to antibiotics in order to identify the strains with high pathogenic potential

Madge - Microplate Array Diagonal Gel Electrophoresis

Microplate array diagonal gel electrophoresis (MADGE) was invented for molecular genetic epidemiological studies. MADGE is a highly flexible, cost effective, microplate compatible solution to high throughput electrophoresis needs. It enables several thousands to million gel lines per day for direct assay of single-base variation in different capacity laboratories. Variants of the standard 96-well MADGE include: 96-well stretch-MADGE, 192-well MADGE, 384-well MADGE, and 768-well MADGE. Melt-MADGE combines the temporal thermal ramp apparatus to achieve similar throughput for de novo mutation scanning. Basic MADGE principles and procedures, preparation of MADGE gels, electrophoresis, visualization and analysis of these gels, as well as modifications of the basic 96-well MADGE will be discussed in detail. For the first time in our country, this revolutionary polyacrylamid electrophoresis was done in 1998. We shortly review our studies which used MADGE for high throughput genotyping of the apolipoprotein E. MADGE and melt-MADGE will have an important role in the future genetic research of complex diseases and especially in pharmacogenomics.5th EFCC Symposium on Proteins - From Electrophoresis to Proteomics, 2009, Belgrade, Serbi

Dietary amino acid and vitamin complex protects honey bee from immunosuppression caused by Nosema ceranae

Microsporidium Nosema ceranae is well known for exerting a negative impact on honey bee health, including down-regulation of immunoregulatory genes. Protein nutrition has been proven to have beneficial effects on bee immunity and other aspects of bee health. Bearing this in mind, the aim of our study was to evaluate the potential of a dietary amino acid and vitamin complex "BEEWELL AminoPlus" to protect honey bees from immunosuppression induced by N. ceranae. In a laboratory experiment bees were infected with N. ceranae and treated with supplement on first, third, sixth and ninth day after emergence. The expression of genes for immune-related peptides (abaecin, apidaecin, hymenoptaecin, defensin and vitellogenin) was compared between groups. The results revealed significantly lower (p lt 0.01 or p lt 0.001) numbers of Nosema spores in supplemented groups than in the control especially on day 12 post infection. With the exception of abacein, the expression levels of immune-related peptides were significantly suppressed (p lt 0.01 or p lt 0.001) in control group on the 12th day post infection, compared to bees that received the supplement. It was supposed that N. ceranae had a negative impact on bee immunity and that the tested amino acid and vitamin complex modified the expression of immune-related genes in honey bees compromised by infection, suggesting immune-stimulation that reflects in the increase in resistance to diseases and reduced bee mortality. The supplement exerted best efficacy when applied simultaneously with Nosema infection, which can help us to assume the most suitable period for its application in the hive

Study of the deamalgamation process in silver-tin-base amalgams

Deamalgamation of the common dentistry amalgam scrap, as well as of a model Ag_70Sn_17Cu_13 prealloy amalgam, was studied by metallography, microhardness, X-ray diffraction and differential thermal analysis techniques. The results are discussed from the view-point of the possible powder-metallurgical application of the process

Supplementary data for article: Stanković, N.; Šenerović, L.; Bojic-Trbojevic, Z.; Vuckovic, I.; Vicovac, L.; Vasiljevic, B.; Nikodinović-Runić, J. Didehydroroflamycoin Pentaene Macrolide Family from Streptomyces Durmitorensis MS405(T): Production Optimization and Antimicrobial Activity. Journal of Applied Microbiology 2013, 115 (6), 1297–1306. https://doi.org/10.1111/jam.12326

Supporting information for: [https://doi.org/10.1111/jam.12326]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1438]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3486

Istraživanje deamalgamacijskog procesa u amalgamima slitina bogatih srebrom i kositrom

Proces deamalgamacije u amalgamima slitina bogatih srebrom i kositrom istraživan je metalografijom, mjerenjem mikrotvrdoće, rentgenskom difrakcijom i diferencijalnom termičkom analizom. Dobiveni rezultati raspravljeni su s gledišta regeneracije otpadnog amalgama (reciklaže, tj. obnavljanja strukture i svojstava polaznih komponenti), kao i moguće uporabe ovog procesa u metalurgiji praha