Structural and functional characterisation of MspA, the main porin of Mycobacterium smegmatis:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | German |
| Veröffentlicht: |
2004
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Erlangen-Nürnberg, Univ., Diss., 2004. - Enth. Zsfassung in dt. Sprache |
| Beschreibung: | IV, 107 S. Ill., graph. Darst. |
Internformat
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Datensatz im Suchindex
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| adam_text | INDEX
1 Summary/Zusammenfassung 1
2 INTRODUCTION 2
2.1 The genus Mycobacterium 2
2.1.1 Classification 2
2.1.2 Medical relevance of mycobacteria 2
2.2 Mycobacterial cell envelope 3
2.3 Porins of Gram negative bacteria S
2.4 Mycobacterial porins 6
2.5 Porins of Mycobacterium smegmatis 7
2.6 Permeability of mycobacterial outer membrane 8
2.7 Porin mutants 9
2.8 Scope of the thesis 10
3 MATERIAL AND METHODS 11
3.1 Materials 11
3.1.1 Chemicals, equipment and oligonucleotides 11
3.1.2 Bacterial strains and plasm ids 14
3.2 Media, buffers and solutions 15
3.2.1 Media 16
3.2.2 Buffers and Solutions 16
3.3 Method 18
3.3.1 General Methods 18
3.3.2 Growth of bacteria 19
3.4 Transformation of bacteria 19
3.4.1 Electroporation of £ coli. 19
3.4.2 Electroporation of M. smegmatis 19
3.4.3 Electroporation of slow growing mycobacteria 19
3.5 Methods for nucleic acid purification and modification 20
3.5.1 Purification of plasmid DNA with modified alkaline / SDS lyses 20
3.5.2 Preparation of chromosomal DNA from mycobacteria 20
3.5.3 Isolation of plasmid DNA from mycobacteria 20
3.5.4 Isolation of DNA from SDS Polyacrylamide gel 20
3.5.5 Polymerase chain reaction (PCR) 20
3.6 Methods for protein extraction, detection and quantification 21
3.6.1 Extraction of the total protein of M. smegmatis 21
3.6.2 Extraction of the MspA protein of M. smegmatis 21
3.6.3 Denaturing Polyacrylamide gel electrophoresis (PAGE) 21
3.6.4 Silver staining of proteins 22
3.6.5 Coomassie brilliant blue staining of proteins 22
3.6.6 Sonification 22
3.6.7 Determination of protein concentration 22
3.6.8 Western Blot analysis 22
3.7 Methods for in vitro mutagenesis 23
3.7.1 Random mutagenesis PCR (error prone PCR) 23
Index ^^ _jj.
3.7.2 DNA shuffling . .23
3* „ .Method8 for alkaline phosphatase assay in inycoi»ctertorZZZ...23
3.8.1 Amplification of the phoA gene and construction of pML440
{PimrWhoA) 23
3.8.2 Alkaline phosphatase assay !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!.. 24
3.9 Growth of M. smegmatis on sugars . . 24
3.10 Computer analyses ZZ!ZZ! 24
3 l 4 Construction of cystelne mutants and label witti maieiniid biocytin !!!25
3.11.1 Site directed mutagenesis by CCR 25
3.11.2 Optimization ofthebiotinylation reaction 25
3.11.3 Biotinytation of MspA cysteine mutants ...25
3.11.3.1 Biotinylation of MspA in vitro 25
3.11.3.2 Biotinylation of MspA in vivo . 25
3.11.3.3 Gel electrophoresis and Western blots I.!!!!!!!!!!!!!!!!!!!!! 26
3.12.4 Lipid bilayer experiments !!!!!!!!!!!!!!!!!!! 26
3.12.5 Accessibility of the cysteine in the crystal structure ofMspA!!!!!!!!!!! 27
4 RESULTS _ 28
4.1 Growth of M. smegmatis on sugars... 28
4.2 In vitro mutagenesis of MspA Z. 33
4.2.1 Error prone PCR 33
4.2.2 DNA shuffling !!!!!!!!!!! ! 35
4.3 Use of PhoA to assay the outer membranepermeability of
mycobactena 35
t l l p°nstruction of M. smegmatis pnoAj^h^on Z ZZ Z ..37
Hi L J.If6 °f the inor9anic Phosphate on the M. smegmatis phoA 39
4.3.3 Phosphatase assay to measure the porin mediated outer membrane
permebihty in mycobactena 40
4.3.3.1 Expression of phoA in M. bows BCG 41
4.3.4 Integration of a phoA expression cassette into the chromosome of
M. smegmatis 42
4.3.4.1 Strategy ZZ Z 42
4.3.4.2 Construction of the recombinant strains .43
.. 4o43 . PnosPnatase activity assay in the recombinant strains 45
4.4 Screening of M. smegmatis for MspA mutants using the phoA
4.4.1 Removal of the FRT fianked hygromycin resisVance cassette from ML10attB::phoA 47
4.4.2 Screening for MspA mutants . . . . . . . . . . . . ^50
4.4.3 Characterisation of plasmids with mutated DNA fragmerits !!!. Z 51
4.4.3.1 Phosphatase activity assay in M. smegmatis mutants 52
4.4.3.2 Expression of the MspA mutants in M. smegmatis mutants 53
4.4.3.3 Analysis of MspA mutants 54
4.5 Construction of MspA cysteine mutants !! ..58
4.6 Expression of MspA cysteine mutants in M.smegmafe!.!!!!!!!.!!!.!!!!!!!!!61
4.7 Biotinylation Of MspA cysteine mutants in Vitro 63
4.8 Optimization of the biotinylation reaction ZZZZZZZ. !64
4.9 Westem blot analysis 66
4.10 Reaction of MspA channel with maleimide biotin !!!Z!!!Z!!!Z.. 70
Index iii
5 DISCUSSION 72
5.1 Carbohydrate utilisation in M. smegmatis —72
5.2 Selection for MspA protein with increased channel size 76
5.2.1 Use of PhoA to assay the OM permeability of mycobacteria 76
5.2.2 Stable integration of a phoA expression cassette in mycobacteria 77
5.2.3 Screening for MspA mutants with increased channel size 78
5.3 Topology of the MspA in the outer membrane of M. smegmatis 79
5.3.1 Most MspA cys mutants are localized in the OM of M. smegmatis 79
5.3.2 Examination of the topology of MspA protein using Biotin containing
sulfhydryi.reagent 80
5.3.3 Effect of maleimide biotin on the channel function of MspA cysteine
mutants 85
6 REFERENCES 87
7 APPENDIX 97
7.1 Analysis of MspA cysteine mutants 97
7.2 Lipid bilayer analysis of the MspA Cys mutants 101
7.3 Abbreviation 105
|
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| author_GND | (DE-588)129721611 |
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| indexdate | 2024-07-09T20:03:35Z |
| institution | BVB |
| language | German |
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| physical | IV, 107 S. Ill., graph. Darst. |
| publishDate | 2004 |
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| spelling | Mahfoud, Maysa 1966- Verfasser (DE-588)129721611 aut Structural and functional characterisation of MspA, the main porin of Mycobacterium smegmatis von Maysa Mahfoud 2004 IV, 107 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Erlangen-Nürnberg, Univ., Diss., 2004. - Enth. Zsfassung in dt. Sprache Erlangen-Nürnberg, Univ., Diss., 2004 Mycobacterium smegmatis - Porin - Struktur-Aktivitäts-Beziehung Mycobacterium smegmatis (DE-588)4607281-0 gnd rswk-swf Permeabilität (DE-588)4173828-7 gnd rswk-swf Porin (DE-588)4221584-5 gnd rswk-swf Struktur-Aktivitäts-Beziehung (DE-588)4183784-8 gnd rswk-swf Plasmamembran (DE-588)4067550-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Mycobacterium smegmatis (DE-588)4607281-0 s Plasmamembran (DE-588)4067550-6 s Permeabilität (DE-588)4173828-7 s Porin (DE-588)4221584-5 s DE-604 Struktur-Aktivitäts-Beziehung (DE-588)4183784-8 s DE-188 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=013005835&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Mahfoud, Maysa 1966- Structural and functional characterisation of MspA, the main porin of Mycobacterium smegmatis Mycobacterium smegmatis - Porin - Struktur-Aktivitäts-Beziehung Mycobacterium smegmatis (DE-588)4607281-0 gnd Permeabilität (DE-588)4173828-7 gnd Porin (DE-588)4221584-5 gnd Struktur-Aktivitäts-Beziehung (DE-588)4183784-8 gnd Plasmamembran (DE-588)4067550-6 gnd |
| subject_GND | (DE-588)4607281-0 (DE-588)4173828-7 (DE-588)4221584-5 (DE-588)4183784-8 (DE-588)4067550-6 (DE-588)4113937-9 |
| title | Structural and functional characterisation of MspA, the main porin of Mycobacterium smegmatis |
| title_auth | Structural and functional characterisation of MspA, the main porin of Mycobacterium smegmatis |
| title_exact_search | Structural and functional characterisation of MspA, the main porin of Mycobacterium smegmatis |
| title_full | Structural and functional characterisation of MspA, the main porin of Mycobacterium smegmatis von Maysa Mahfoud |
| title_fullStr | Structural and functional characterisation of MspA, the main porin of Mycobacterium smegmatis von Maysa Mahfoud |
| title_full_unstemmed | Structural and functional characterisation of MspA, the main porin of Mycobacterium smegmatis von Maysa Mahfoud |
| title_short | Structural and functional characterisation of MspA, the main porin of Mycobacterium smegmatis |
| title_sort | structural and functional characterisation of mspa the main porin of mycobacterium smegmatis |
| topic | Mycobacterium smegmatis - Porin - Struktur-Aktivitäts-Beziehung Mycobacterium smegmatis (DE-588)4607281-0 gnd Permeabilität (DE-588)4173828-7 gnd Porin (DE-588)4221584-5 gnd Struktur-Aktivitäts-Beziehung (DE-588)4183784-8 gnd Plasmamembran (DE-588)4067550-6 gnd |
| topic_facet | Mycobacterium smegmatis - Porin - Struktur-Aktivitäts-Beziehung Mycobacterium smegmatis Permeabilität Porin Struktur-Aktivitäts-Beziehung Plasmamembran Hochschulschrift |
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