PROGRAM OF VIII SUMMER SCHOOL
«MOLECULAR MICROBIOLOGY AND BIOTECHNOLOGY»
Оdessa, 27 May – 14 June 2013
Theoretical course (46 hours)
Molecular genetics and molecular microbiology. Modern scientific concepts in molecular microbiology. Biological metronome. Theory of Neutral evolution. Adaptive mutations. The Prokaryote-Eukaryote dichotomy. Modern schemes in classification of Prokaryote and Eukaryote microorganisms and viruses. Domains of life (2 hours).
Lecture 2 and 3
Primary genomes sequence. Methods of primary sequence identification – sequencing. Bacterial genomics. Topology of total bacterial genomes. Orthologs and parologs. Clusters of orthologous genes (COG). The concept of “the smallest” genome. (4 hours).
Bacterial autonomous genetic elements. Insertion sequences, transposons, restriction-modification systems, plasmids, bacteriophages, pathogenicity islets and islands. Hierarchy and mobility of autonomous genetic elements. Autonomous genetic elements and emergence of new pathogenic bacteria. (2 hours).
Bacterial viruses. Modern classification of bacteriophages. Tailed phages– order Caudovirales. Temperate and virulent bacteriophages. The strategy of caudoviruses development. Bacterial and Archaea viruses. Phages of enterobacteria and lactic acid bacteria. Phage therapy. (2 hours).
The genomics of prophages and pathogenisity islands. Phage lysogenic conversion. Phage toxins. Concept of “morones”. Pathogeny islands and type III secretion system. (2 hours).
Defective bacteriophages and. The role in the interbacterial antagonism. The perspectives of bacteriocines usage: typology of bacteria, biocontrol of bacterial populations and cancer therapy. (2 hours).
Plasmid genomics, Plasmid determinants of pathogenicity. Plasmids based vectors. Molecular genomics of plasmids pTi – C58. Agrobacterium tumefaciens. Transgenic plants. (2 hours).
Bacterial transposons, IS-elements, integrones and gene cassettes. Formation of bacterial multiple antibiotic resistance. Concept of inner genome transformations – genome engineering. (2 hours).
Ecology of bacterial viruses. Distribution and role in global metabolism. New methods in bacteriophages ecology. The World Ocean and virioplancton. (2 hours).
Cluster analysis. Evaluation of objects kinship. The problem of kinship measure adequacy and characteristics of organisms kinship. (2 hours).
Cluster analyses methods. Direct classification algorithms. Clusters determination. Cluster analysis measuring algorithms used in biology. The criteria for classification quality. (2 hours)
Numeric taxonomy. Hierarchical algorithms. The use of cluster analyses in numeric taxonomy and phylogeny. (2 hours).
Lectures 1-13 (26 hours) by Members of Institute of Microbiology and Virology NASU (Prof. Tovkach F. I.) and Odessa Mechnykov National University.
Lecture 14-23 (20 hours) VNII of agr. microbiology and others.
Tikhonovich Y.A. Epispecies genetic systems (2 hours)
Provorov N.A. Bacterial evolution in the microbe-plant symbiosis system (2 hours)
Borysov A.U. Legumes and microorganisms mutualisms: genetics, evolution, practical use. (2 hours)
Andronov E.E. Metagenomics of soil microorganisms community (2 hours)
Chebotar V.K. Endophytic bacteria of higher plants: role in host development and perspectives for use (2 hours)
Onischuk O.P. Ecologic genetics of nodule-forming bacteria. (2 hours)
Belimov A.A. Plants hormone status mediation by microorganisms in stress conditions (2 hours)
Lugova L.A. (St. Petersburg state university) Molecular genetic aspects and modern methods of plant development biology (2 hours)
Gogolev U.V. (University of Kazan) Bacterial anti-sense regulatory RNA (2 hours)
Kamnev A.A. (IBFRM RAN, Saratov) Modern infrared spectroscopy in microbial ecology: methodology and use (2 hours)
Practical course (70 hours)
Laboratory lesson 1
Phages of order Caudovirales. Bacteriophages of Escherichia coli: Т7 (family Podoviridae), lambda (Syphoviridae), Р1 and Т4 (Myoviridae). Classification. Моrphotypes, organization of virions. Virus DNA.
Тitration of phages. Phage plaques, their accordance to morphotype, virion size and the nature of the viruses.
Preparative obtaining of phage particles. The method of fuse lysis of susceptible culture. Concentration of phage particles by PEG-precipitation method (Yamamoto procedure).
Isolation of viral DNA. Rules of work with DNA.
Restriction analysis. Identification of bacterial viruses by restriction patterns of their genomes. (ONU, Sergeeva Zh.U., Krylova K.D., Ivanytsia T.V.)
Laboratory lesson 2
Molecular genetics of temperate coliphage P1. Common and specialized P1-transduction of genetic markers. Plasmid prophage. Phage system of restriction-modification ЕсоР1.
Temperature induction of lysogen E. coli С600 [P1::Tn9 (Cmсts 100)].
Obtaining and testing of Р1-lysogens.
Plasmid profiles of lysogens.
Limitation of lambda bacteriophage productive development in cells of E. coli (Р1) owing to R/М system ЕсоР1.
Lysogenization of Erwinia carotovora cells by phage P1::Tn9.
Incorporation of Tn9 transposone into cryptic plasmid рСА25 of E. carotovora. (ONU, Sergeeva Zh.U., Krylova K.D., Ivanytsia T.V.)
Laboratory lesson 3
Plasmid profiles of bacteria. Form and size of nonchromosomal circled DNA.
Peculiarities of plasmid isolation in gramnegative bacteria from natural ecological niches. Alkaline method of Kado and Liu.
Isolation of plasmid prophage P1 and enterobacterial plasmids F, RP4, pCA25, рСА25::Tn9. Electrophoretic partition of plasmid DNA. Electrophoretic mobility of DNA depends on its size.
Transformation of bacterial cells (method using calcium chloride) (ONU, Sergeeva Zh.U., Krylova K.D., Ivanytsia T.V.)
Ion Exchange and Size Exclusion Chromatography of virions and their components.
Principles of liquid chromatography. Chromatography of high and low pressure.
Modern equipment for chromatography. Isolation of phage P1 and T7 virions. Analysis of elution profiles. Electrophoresis in polyacrylamide gels. (BIO-RAD, Gyula Csanadi, IMV, Ostapchuk A.M.)
Laboratory lesson 5
Polymerase chain reaction (PCR) – synthesis of certain DNA fragment invitro. Types of PCR. PCR-diagnostics of crown gall of grape: revealing of pTi sequences of pathogenic strains of Agrobacterium tumefaciens and Agrobacterium vitis. “Bio-PCR” method in plant disease diagnostics. PCR-laboratory.
Isolation of plasmid DNA by heat lysis of bacterial cells. Reaction mixes for PCR.
Amplification of pTi sequences of Agrobacterium tumefaciens and Agrobacterium vitis strains.
Electrophoresis of PCR products. (ONU, Limanska N.V.)
Laboratory lesson 6
Use of cluster analysis in revealing of relationships. Creation of phylograms using computer programs.
Plural equalization as a preliminary stage in analysis of relationships.
Common program packets used in phylogeny (programs on-line).
Program f or primary sequence analysis and creation phylograms using this program (packet MatLab). (ONU, Vasyleva N.U.)