Anaerobic pathways in the porifera [Elektronische Ressource] : strombine dehydrogenase, an opine dehydrogenase, from the sponge Suberites domuncula / Bruna Pleše

Bruna

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2007
• Nombre de lectures: 30
• Poids de l'ouvrage: 2 Mo

Extrait

Anaerobic pathways in the Porifera: Strombine
dehydrogenase, an opine dehydrogenase, from the sponge
Suberites domuncula








Dissertation
zur Erlangung des Grades
Doktor der Naturwissenschaften




Am Fachbereich Biologie
der Johannes Gutenberg-Universität
in Mainz








Bruna Pleše
geb. in Zagreb (Croatia)








Mainz, 2007







































Dekan:
1. Berichterstatter:
2. Berichterstatter:
Tag der mündlichen Prüfung:











Mojoj Baki i Čiki
























1 INTRODUCTION .........................................................................................................1

1.1 Sponges (Porifera)................................................................................................1
1.1.1 The origins ...........................................................................................................1
1.1.2 Morphology...........................................................................................................4
1.1.3 Reproduction.........................................................................................................5
1.1.4 Classification.........................................................................................................6
1.1.5 Symbiosis..............................................................................................................7
1.2 Anaerobic pathways............................................................................................10
1.2.1 Evolution of anaerobic pathways........................................................................12
1.2.2 Opines.................................................................................................................14

2 AIM OF THE STUDY...................................................................................................18

3 MATERIALS AND METHODS...................................................................................19
3.1 Materials.............................................................................................................19
3.1.1 Chemicals............................................................................................................19
3.1.2 Equipment...........................................................................................................20
3.1.3 Enzymes and Antibodies.....................................................................................21
3.1.4 Markers...............................................................................................................21
3.1.5 Kits......................................................................................................................22
3.1.6 Vectors................................................................................................................22
3.1.7 Primers
3.1.8 Bacterial strains...................................................................................................23
3.1.9 Culture medium..................................................................................................24
3.1.10 Computer programs............................................................................................25
3.2 Experimental animals..........................................................................................25
4 Methods ..............................................................................................................26
4.1 DNA extraction from tissue................................................................................26
4.2 Phenol-chloroform DNA extraction ..................................................................27
4.3 DNA precipitation...............................................................................................27
4.4 Restriction digestion of DNA .............................................................................27
4.5 RNA isolation.....................................................................................................28
4.5.1 RNAse inactivation using DEPC (diethylpyrocarbonate) ................................28
4.6 Southern Blotting ...............................................................................................28
4.7 Screening of the S. domuncula genomic library .................................................30
4.7.1 Plating out the library and transferring the lambda phage to nylon membranes 32
4.7.2 Lambda DNA extraction.....................................................................................33
4.8 Isolation of plasmid DNA ..................................................................................34
4.9 Agarose gel electrophoresis ..............................................................................34
4.10 Isolation of DNA from agarose gel.....................................................................35
4.11 Polyacrylamide gel..............................................................................................35
4.12 Photometric measurements.................................................................................36
4.13 Cloning................................................................................................................36
4.13.1 A-Tailing of DNA for TA ligation and cloning..................................................36
4.13.2 TOPO TA cloning...............................................................................................37
4.13.3 pGEM-T cloning.................................................................................................37
4.14 Transformantion of competent cells .................................................................38
4.15 Optic density (OD) of bacterial cultures.............................................................40
4.16 Generation of primers.........................................................................................40
4.17 PCR (Polymerase Chain Reaction).....................................................................41
4.17.1 Long and accurate (LA) PCR .............................................................................42
4.17.2 Touch down PCR................................................................................................43
4.17.3 DIG labeling PCR...............................................................................................43
4.17.4 Checking PCR.....................................................................................................45
4.17.5 Semi-quantitative RT-PCR.................................................................................45
4.18. DNA Sequencing................................................................................................46
4.19 Sequence analysis ...............................................................................................47
4.20 Isolation of total protein extract..........................................................................48
4.21 SDS-polyacrylamid gel electrophoresis (SDS-PAGE).......................................48
4.21.1 Native/Seminative PAGE ...................................................................................50
®4.21.2 Coomassie staining with GelCode Blue stain reagent ......................................51
4.21.3 Measuring protein concentration (Bradford) ......................................................51
4.21.3.1 Protein Standards ................................................................................................51
4.22 Western Blot.......................................................................................................52
4.23 Recombinant His-tag fusion protein ...................................................................53
4.23.1 Expression of His-tag fusion protein ..................................................................53
4.23.2 Determination of target protein solubility...........................................................54
4.23.3 Purification of 6xHis-tagged fusion proteins using Ni-NTA spin columns .......55
4.24 Antibody production ...........................................................................................57
4.25 Immunohistochemistry.......................................................................................58
4.26 ELISA (Enzyme Linked Immunosorbent Assay)...............................................59
4.27 Enzyme assays ....................................................................................................61
4.27.1 Substrate Saturation of the Enzyme....................................................................63
4.28 Protein modeling......................................................................