SULFOLOBUS SOLFATARICUS AS SOURCE OF GLYCOSYL HYDROLASES WITH PDF
Preview SULFOLOBUS SOLFATARICUS AS SOURCE OF GLYCOSYL HYDROLASES WITH
SSUULLFFOOLLOOBBUUSS SSOOLLFFAATTAARRIICCUUSS AASS SSOOUURRCCEE OOFF GGLLYYCCOOSSYYLL HHYYDDRROOLLAASSEESS WWIITTHH BBIIOOTTEECCHHNNOOLLOOGGIICCAALL PPOOTTEENNTTIIAALL Michele Girfoglio Dottorato in Scienze Biotecnologiche – XXI ciclo Indirizzo Biotecnologie Industriali Università di Napoli Federico II Dottorato in Scienze Biotecnologiche – XXI ciclo Indirizzo Biotecnologie Industriali Università di Napoli Federico II SSUULLFFOOLLOOBBUUSS SSOOLLFFAATTAARRIICCUUSS AASS SSOOUURRCCEE OOFF GGLLYYCCOOSSYYLL HHYYDDRROOLLAASSEESS WWIITTHH BBIIOOTTEECCHHNNOOLLOOGGIICCAALL PPOOTTEENNTTIIAALL Michele Girfoglio Dottorando: Michele Girfoglio Relatore: Prof. Mosè Rossi Coordinatore: Prof. Giovanni Sannia A tutte le persone a me care, che mi hanno sempre sostenuto ed hanno rappresentato un fondamentale supporto durante il percorso di cui questa tesi rappresenta il compimento. A Elia P. INDEX ABBREVIATIONS pag. 1 RIASSUNTO pag. 3 SUMMARY pag. 10 INTRODUCTION pag. 12 Archaea pag. 12 Sulfolobus pag. 13 Genetic elements pag. 14 Plasmid-fusellovirus genetic systems pag. 20 S. solfataricus-E. coli shuttle vectors pag. 22 Thermophilic enzymes as biocatalysts in industrial applications pag. 24 Cellulose pag. 24 Concepts, methodology and objectives pag. 28 MATERIAL AND METHODS pag. 30 Strains, enzymes and reagents used in this study pag. 30 E. coli transformation techniques pag. 30 Proteins analyses pag. 31 Cloning of the lacS gene into pMSSV and characterization of the vector pag. 32 sso1354 gene expression in S. solfataricus pag. 35 sso1354 gene expression in E. coli pag. 37 sso1354 gene expression in K. lactis pag. 38 Enzyme assays and analyses pag. 38 RESULTS pag. 40 1. CHARACTERIZATION OF pMSSV SHUTTLE VECTOR: CLONING AND pag. 40 EXPRESSION OF THE lacs GENE Cloning of the expression cassette tf55αlacS in the pMSSV vector pag. 40 lacS expression in the β-galactosidase deficient mutant GθW pag. 41 β-galactosidase assays on pMSSVlacS transfectants pag. 44 2. XENOLOGOUS AND HETEROLOGOUS EXPRESSION OF sso1354 GENE pag. 45 Sequence analysis pag. 45 Cloning of the expression cassette araS1354 in the pMSSV vector and pag. 49 expression in S. solfataricus Expression of the sso1354 gene in E. coli pag. 53 Expression and characterization of the sso1354 gene in Kluyveromyces pag. 54 lactis DISCUSSION pag. 57 ABBREVIATIONS bp Base pairs BSA Bovine serum albuine CAPS N-cyclohexyl-3-aminopropanesulfonic acid CBD Cellulose binding domain CPs Conjugative plasmids Da dalton DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate DSM Deutsche Sammlung von Mikroorganismen EDTA Ethylenediaminetetraacetic acid h Hours HGT Horizontal gene transfer hph Hygromycin B phosphotransferase IPTG Isopropyl β-D-1-thiogalactopyranoside IS Insertion sequence LB Luria Bertani medium min Minutes MITEs Miniature inverted transposable elements OD Optical density ORF Open reading frame PCR Polimerase chain reaction PMSF Phenyl methane sulphonyl fluoride PVDF Polyvinylidene difluoride RNA Ribonucleic acid 1 SBP Sugar binding protein SDS Sodium Dodecyl Sulphate SDS-PAGE Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis TBE 45 mM Tris-borate, 1mM EDTA pH 8.0 TBP TATA-binding protein TLC Thin Layer Chromatography Tris tris(hydroxymethyl)aminomethane TYS tryptone yeast sucrose (medium) UTR untraslated region X-gal 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside YPG Yeast extract Peptone Galactose 2
Description:The list of books you might like
