Genomic analysis of the lysogeny module of temperate Streptococcus thermophilus bacteriophage TP-J34 [Elektronische Ressource] / vorgelegt von Mazhar Desouki Ali Mohamed

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Informations

• Publié par: christian-albrechts-universitat_zu_kiel
• Publié le: 01 janvier 2010
• Nombre de lectures: 42
• Langue: Deutsch
• Poids de l'ouvrage: 13 Mo

Extrait

Aus dem Institut für Mikrobiologie und Biotechnologie
Max Rubner-Institut
Bundesforschungsinstitut für Ernährung und Lebensmittel





Genomic Analysis of the Lysogeny
Module
of Temperate Streptococcus
thermophilus
Bacteriophage TP-J34


Dissertation
zur Erlangung des Doktorgrades
der Agrar- und Ernährungswissenschaftlichen Fakultät
der Christian-Albrechts-Universität zu Kiel


vorgelegt von

M.Sc. Mazhar Desouki Ali Mohamed
aus Sohag, Ägypten



Kiel, 2010

Dekanin: Prof. Dr. Karin Schwarz
Erster Berichterstatter: Prof. Dr. Knut J. Heller
Zweiter Berichterstatter: Prof. Dr. Frank Döring
Tag der mündlichen Prüfung: 10th February .2011





















„Gedruckt mit Genehmigung der Agrar- und Ernährungswissenschaftlichen
Fakultät der Christian-Albrechts-Universität zu Kiel“










Table of Contents
Table of Contents.................................................................... IV
1 Chapter 1: ..............................................................................1
Introduction .................................................................................1
1.1 Bacteriophages.......................................................................................................1
1.1.1 Composition and classification of bacteriophage.......................................3
1.1.2 Life cycle of temperate phage ..........................................................................4
1.1.3 Gene regulation and genetic switch in phage lambda ............................7
1.2 LAB.............................................................................................................................11
1.3 Bacteriophage infection of LAB........................................................................12
1.4 Streptococcus thermophilus..............................................................................13
1.5 Phages of S. thermophilus ..................................................................................14
1.6 Temperate S. thermophilus phage TP-J34 .....................................................15
1.6.1 The lysogeny module of phage TP-J34...........................................................17
1.6.2 The genetic switch in TP-J34 ..............................................................................19
1.7 The aim of this work..............................................................................................20
2 Chapter 2: ............................................................................22
Materials and Methods..........................................................22
2.1 Bacterial cultures and growth conditions ......................................................22
2.2 Polymerase chain reaction (PCR)....................................................................23
2.2.1 Colony PCR.............................................................................................................23
2.2.2 Primer design..........................................................................................................24
2.2.3 Designing of PCR programs...............................................................................25
2.3 Induction of prophage using mitomycin C (MC) ........................................25
2.4 Isolation of chromosomal DNA of S. thermophilus......................................26
2.4.1 Reagents .................................................................................................................26
2.4.2 Procedure ...............................................................................................................26
2.5 Agarose gel electrophoresis .............................................................................28
2.5.1 Reagents .................................................................................................................28
2.5.2 Procedure ...............................................................................................................28
2.6 Plasmid DNA isolation..........................................................................................29
2.6.1 Isolation from E. coli..............................................................................................29
2.6.2 Isolation from S. thermophilus............................................................................30
2.7 Purification of PCR product from agarose gel ..............................................32
Mazhar Mohamed V

Table of Contents


2.8 Preparation of E. coli EC1000 competent cells ............................................32
2.9 Preparation of S. thermophilus competent cells..........................................33
2.10 Cloning.....................................................................................................................33
2.10.1 Dephosphorylation of insert DNA.....................................................................33
2.10.2 Cloning of an internal fragment of int into pGhost9 (pMAZ2).................34
2.10.3 Cloning of orf3 in the expression vector pMG36e.......................................34
2.11 Electro-transformation of E. coli EC1000.........................................................35
2.12 Electro-transformation of S. thermophilus J34f-2..........................................36
2.13 Integration of pMAZ2 into chromosomal DNA of S. thermophilus
J34f-2 ........................................................................................................................36
2.14 Isolation of a mutation in orf3............................................................................37
2.15 DNA sequencing...................................................................................................38
2.16 Analysis of mutated orf3 gene..........................................................................38
2.17 Sequencing of RT-PCR products within pSTBlue-1 .......................................38
2.18 Southern blot analysis..........................................................................................40
2.18.1 Procedure ...............................................................................................................41
2.18.2 Reagents .................................................................................................................44
2.19 RNA manipulations...............................................................................................47
2.19.1 Calculation of the amount of bacterial culture for RNA isolation..........48
2.19.2 RNase Decontamination ....................................................................................48
2.19.3 Protection of cellular RNA prior to isolation...................................................49
2.19.4 Isolation of RNA .....................................................................................................50
2.19.5 Purification of RNA................................................................................................51
2.19.6 Removing contaminating genomic DNA......................................................52
2.19.7 Detection of RNA integrity and purity.............................................................53
2.19.8 Quantification of RNA solutions ........................................................................54
2.19.9 Preparation of acid washed glass beads (for RNA isolation) ..................54
2.20 Northern blot...........................................................................................................55
2.20.1 Reagents .................................................................................................................55
2.20.2 Procedure ...............................................................................................................55
2.21 Double-stranded DNA probe ............................................................................57
2.21.1 DIG labeling DNA probes ...................................................................................57
2.21.2 Determination of labeling efficiency of probes...........................................58
2.21.3 Preparation of probe solution for use .............................................................60
2.21.4 Storage and reuse of hybridization solution..................................................60
2.22 Reverse transcription PCR (RT-PCR) .................................................................60
2.22.1 Reactions solution.................................................................................................60
2.22.2 RT-PCR programs...................................................................................................61
2.23 Media and buffers .............................................