PROGRAM OF VII SUMMER SCHOOL
«MOLECULAR MICROBIOLOGY AND BIOTECHNOLOGY»
Оdessa, 5 June – 20 June 2012
Theoretical course (20 hours)- Biosensors
Practical course (80 hours)
Laboratory lesson 1
Phages of order Caudovirales. Bacteriophages Escherichiacoli: 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.
Laboratory lesson 2
Molecular genetics of temperate coliphage P1. Common and specialized P1-transduction of genetic markers. Plasmid prophage. Phage system of restriction-modification ЕсоР.
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/Мsystem ЕсоР1.
Lysogenization of Erwiniacarotovora cells by phage P1::Tn9.
Incorporation of Tn9 transposone into cryptic plasmid рСА25 of E.carotovora.
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, рСА25::Tn9. Electrophoretic partition of plasmid DNA. Electrophoretic mobility of DNA depends on its size.
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.
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 f or primary sequence analysis and creation phylograms using this program (packet MatLab).