THE PROGRAMME OF IV SUMMER SCHOOL
«MOLECULAR MICROBIOLOGY AND BIOTECHNOLOGY»
Odessa, 4th – 16th of May 2009
- Odesa I.I. Mechnykov National University of MES of Ukraine
- Zabolotny Microbiology and Virology Institute of NAS of Ukraine
- Winogradsky Society of Ukrainian microbiologists
Autonomic genetic elements of bacteria: bacteriophages, plasmids and transposones
Theoretical course (20 hours)
Molecular genetics and molecular microbiology. Modern scientific conceptions in molecular microbiology. Biological metronome. The theory of neutral evolution. Adaptive mutations. Procaryotic-eukaryotic dichotomy. Modern classification schemes of prokaryotic and eukaryotic microorganisms and viruses. Domains of life.
Lections 2 and 3
Primary genome sequences. Methods of primary genome sequences revealing – sequence. Bacterial genomes. Topology of integral bacterial genomes. Orthologs and paralogs. Clusters of orthological genes. Conception of the “least” genome.
Autonomic genetic elements of bacteria. Transposones, systems of restriction-modification, plasmids, bacteriophages, pathogenecity islands. Hierarchy and mobility of autonomic genetic elements. Autonomic genetic elements and origin of new pathogenic bacteria.
Bacterial viruses. Modern classification of bacteriophages. Phages with caudal appendixes – Order Caudovirales. Temperate and virulent bacteriophages. Strategy of caudoviruses development. Viruses of bacteria and archaebacteria. Phages of enterobacteria and lactic acid bacteria. Phagotherapy.
Genomics of prophages and pathogenecity islands. Phage lysogenic conversion. Phage toxins. Conceptions of “morons”. Pathogenecity islands and secretion system of III type.
Defective bacteriophages and bacteriocins. Their role in interbacterial antagonism. The perspectives of bactericins use – typing of bacteria, biocontrol of bacterial populations and cancer therapy.
Genomics of plasmids. Plasmid determinants of pathogenecity. Plasmid-based vectors. Molecular genetics of pTi – C58 Agrobacteriumtumifaciens. Transgenic plants.
Bacterial transposones, IS-elements, integrones and genetic cassets. Formation of multiresistance against antibiotics in bacteria. Conception of intragenomic rebuilding – genomic engineering.
Ecology of bacterial viruses. Their spread and role in global substance exchanges. New methods in bacteriophage ecology. World Ocean and virioplanktone.
Cluster analysis. Measuring of the objects relationships. The problem of adequate characteristics organisms relationships.
Cluster analysis methods. Algorithms of direct classification. Determination of clusters. Algorithms of cluster analysis calculation used in biology. Criteria of classification quality.
Numeric taxonomy. Hierarchic algorithms. Use of cluster analysis in numeric taxonomy and phylogeny.
Practical course (60 hours)
Laboratory lesson 1
Phages of order Caudovirales. Bacteriophages Escherichiacoli:(family Podoviridae), lambda (Syphoviridae), (Myoviridae). Classification of Оrphotypes, organization of virions. DNA Virus.
- 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.
Laboratory lesson 2
Molecular genetics of temperate coliphage P1. Common and specialized P1-transduction of genetic markers. Plasmid prophage. Phage system of restriction-modification.
- 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.
- Lysogenization of Erwiniacarotovora cells by phage P1::Tn9.
- Incorporation of Tn9 transposone into cryptic plasmid.
Laboratory lesson 3
Plasmid profiles of bacteria. Form and size of nonchromosomal circled DNA.
- Pecularities 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. Electrophoretic partition of plasmid DNA. Electrophoretic mobility of DNA depends on its size.
Laboratory lesson 4
Creation of conditions for horizontal plasmid transfer into bacterial cells. Plasmid vectors.
- Тransconjugation plasmids. Transfer of plasmid R68.45 from E. coliinto E.carotovora. Selection of recipient and contrselection of donor. The frequency of conjugation.
- Obtaining of calcium-competent cells. Plasmid DNA isolation.
Transformation of E. coli DH and JM109 by Blue plasmids pUC 18 и pBluescript. The frequency of transformation.
- Plasmid profiles of transconjugants and transformants.
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 Agrobacteriumtumefaciens and Agrobacteriumvitis. “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 Agrobacteriumtumefaciens and Agrobacteriumvitisstrains.
- Electrophoresis of PCR products.
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 of primary sequence analysis and creation phylograms using this program (packet MatLab).
Ph.D. in biological sciences,
the chief of Bacteriophage Molecular
Biology Department, professor of
Microbiology and Virology Chair of ONU, F.I. Tovkach