Porins in the genus Borrelia [Elektronische Ressource] : characterization of P66 and P13 = Porins in der Gattung Borrelia : Charakterizierung von P13 und P66 / Iván Bárcena Uribarri

Iván Bárcena Uribarri

Sujets

• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Porins in der Gattung Borrelia,
Charakterizierung von P13 und P66
Porins in the genus
Borrelia
Characterization of P66 and P13








Iván Bárcena Uribarri
from Santander
Graduate School of Life Sciences – University of Wuerzburg
Infection and Immunity
October 2010













Submitted on: …………………………………………………………..……...
Office stamp

Members of the Thesis Committee:
Chairperson: Prof. Dr. Jörg Schultz
Primary Supervisor: Prof. Dr. Roland Benz
Supervisor (Second): Prof. Dr. Thomas Rudel
Supervisor (Third): Prof. Dr. Miquel Viñas
Date of Public Defense: …………………………………………….………….
















I hereby declare that my thesis entitled “Porin in the
genus Borrelia. Characterization from P13 and P66” is the result of
my own work. I did not receive any help or support from commercial
consultants. All sources and/or materials applied are listed and
specified in the thesis.
Furthermore, I verify that this thesis has not yet been
submitted as part of another examination process neither in identical
nor in similar form.


Wuerzburg, October 2010.















Nere amari eskeinita.
Bere ahalegin eta ekimenik gabe, bere babesa eta ulermenik
gabe, tesi hau ez litzaidake posible izango. Momentu onetan
eta batez ere zailtasun uneetan, nere alboan egoteagatik eta
laguntzeagatik.
Maite zaitut, zure.




Publications




 Bárcena-Uribarri* I, Thein* M, Sacher A, Bunikis I, Bonde M, Bergström S, Benz R. P66
porins are present in both Lyme disease and relapsing fever spirochetes: a comparison
of the biophysical properties of P66 porins from six Borrelia species. Biochim Biophys
Acta. 2010 Jun;1798(6):1197-203. Epub 2010 Feb 25.

 Thein* M, Bárcena-Uribarri* I, Maier E, Bonde M, Bunikis I, Bergström S and Benz R.
Use of non-electrolytes reveals the channel size and oligomeric structure of the
Borrelia burgdorferi P66 porin. Biophysical Journal (Submitted).

*both authors contributed equally.



Index
Index
Publications ............................................................................................................................... - 7 -
Abstract ................................... - 13 -
Introduction ............................................................................................................................ - 22 -
1.1 The genus Borrelia ......................................... - 22 -
1.1.1 Characteristics ........................................................................ - 22 -
1.1.2 Classification ........................................... - 23 -
1.2 Human infection ............................................................................ - 23 -
1.2.1 Lyme disease and Relapsing Fever symptoms ....................................................... - 23 -
1.2.2 Lyme disease species and relapsing fever species ................. - 25 -
1.2.3 History of Lyme disease and relapsing fever. ......................................................... - 25 -
1.3 Vectors and Hosts. ........................................................................................................ - 27 -
1.3.1 Borrelia life cycle: ... - 27 -
1.3.2 Borrelia world distribution ..................................................................................... - 29 -
1.4 Outer membrane in Borrelia ......................... - 30 -
1.5 Porins in Gram negative bacteria and porins in Borrelia .............................................. - 31 -
1.5.1 Porins in Borrelia .................................................................... - 32 -
Methods .................................................................. - 33 -
2.1 Borrelia outer membrane isolation ............................................... - 33 -
2.2 Fast Performance Liquid Chromatography ................................... - 34 -
2.3 Wessel-Flügge protein precipitation ............................................. - 34 -
2.4 SDS-Page ........................................................................................ - 35 -
2.5 BN-Page and second dimension SDS page .................................... - 36 -
2.6 SDS-Page silver nitrate staining and gel drying ............................................................. - 37 -
2.7 Black Lipid Bilayer assay ................................................................ - 37 -
2.7.1 Single channel measurements ............................................................................... - 42 -
- 9 -
Index
2.7.2 Ion selectivity ......................................................................................................... - 43 -
2.7.3 Voltage dependency ............................... - 44 -
2.7.4 Substrate specificity ................................................................ - 45 -
2.7.5 Noise analysis. ........................................ - 46 -
2.7.6 Channel diameter determination using non-electrolytes ...... - 47 -
2.8 Western-Blot ................................................................................................................. - 50 -
2.9 Gen cloning .................... - 51 -
2.9.1 P13 cloning outline ................................................................................................. - 52 -
2.9.2 Constructs and primer design ................................................................................ - 54 -
2.9.3 Polymerase Chain Reaction .................... - 55 -
2.9.4 Agarose gel electrophoresis and agarose gel elution ............................................ - 56 -
2.9.5 Vector cloning ........................................................................ - 56 -
2.9.6 Over Night Cultures and type cultures ................................... - 58 -
2.9.7 Plasmid Purification ................................................................ - 59 -
2.9.8 Digestion with restriction enzymes ........................................ - 59 -
2.9.9 Ligation ................................................................................... - 60 -
2.9.10 Transformation ..................................................................................................... - 60 -
2.11 Expression in Escherichia coli ...................................................................................... - 61 -
2.11.1 Expression outline in E. coli .................. - 62 -
2.11.2 Protein induction .................................................................................................. - 63 -
2.11.3 Protein extraction in Escherichia coli. .. - 63 -
2.12 Expression in Nicotiana benthamiana ........................................................................ - 64 -
2.12.1 Agrobacterium infiltration and virus expression.................. - 64 -
2.12.2 Protein extraction in Nicotiana benthamiana...................................................... - 65 -
2. 13 His-tag purification with Ni-NTA resins ...................................................................... - 65 -
Biophysical characterization of P66 in Lyme disease and relapsing fever species ................. - 67 -
3.1 Results ........................................................................................................................... - 68 -
3.1.1 LD and RF P66 gene sequence comparison ............................ - 68 -
- 10 -