5 The role of quorum-sensing in the virulence of Pseudomonas ...

2 Material and Methods
2 MATERIAL AND METHODS 31
GENERAL INFORMATION 32
2.1.1 PREPARATION OF MEDIA 32
2.1.2 STRAINS USED 33
2.1.3 PREPARATION OF ELECTROCOMPETENT CELLS 35
2.1.4 PREPARATION OF CHEMICAL COMPETENT CELLS 36
2.1.5 BACTERIA PLASMID PREPARATION 36
2.1.6 YEAST PLASMID PREPARATION 36
2.1.7 RESTRICTION DIGESTION 37
2.1.8 GEL ELECTROPHORESIS 37
2.2 CONSTRUCTING A CAPTURE VECTOR 38
2.2.1 THE PRINCIPLES OF THE CREATION OF CAPTURE VECTOR 38
2.2.2 PCR PRIMER LIST 38
2.2.3 PRIMER DESIGN FOR GENOMIC WORK 40
2.2.4 AMPLIFICATION THE PLLX8 AMPLICON 41
2.2.5 AMPLIFICATION OF THE TARGETING SEQUENCES, TS1 AND TS2 41
2.2.6 LITHIUM ACETATE TRANSFORMATION 41
2.2.7 SELECTION OF CAPTURE VECTOR 42
2.3 CAPTURING A GENOMIC ISLAND 43
2.3.1 PREPARATION OF HIGH MOLECULAR WEIGHT GENOMIC DNA 43
2.3.2 PREPARATION OF SPHEROPLASTS 43
2.3.3 SCREENING OF SPHEROPLAST COLONIES: COLONY PCR 44
2.3.4 ELECTROPORATION: TRANSFER OF SUSPECT PLASMIDS TO E. COLI 45
2.3.5 RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) 45
2.3.6 SUB-CLONING OF E106-SERW 45
2.4 PREPARATION OF PSEUDOMONAS AERUGINOSA FOR IN VIVO WORK 46
2.4.1 PREPARATION OF BACTERIAL STOCKS 46
2.4.2 INFECTION DOSE PREPARATION 47
2.5 IN VIVO MICE PROTOCOLS 47
2.5.1 ADMINISTRATION 47
2.5.2 ORGAN RETRIEVAL & CFU COUNTS 48
2.5.3 SYMPTOM SCORING 48
2.5.4 HISTOPATHOLOGY 48
2.5.5 LEUKOCYTE COUNTS 49
2.5.6 STATISTICS 50
31