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Chapter 2 47F-60.2-60.1 pre cpn60.2 571-591 48F-60.2-60.1 pre cpn60.2 1126-1146 49R-60.2-60.1 pre cpn60.2 573-553 50R-60.2-60.1 pre cpn60.2 1128-1108 51F-60.1-60.2 pre cpn60.1 571-591 52F-60.1-60.2 pre cpn60.1 1126-1146 53R-60.1-60.2 pre cpn60.1 573-553 54R-60.1-60.2 pre cpn60.1 1128-1108 TTC ACC GAG GGT ATG CGG TTC GAC AAG GGC TAC GGC GGG GTT GCT GTG ATC AAG GCC GGT GCC GCC CTT GTC GAA GCC CAT ACC CTC GGT GAG CTC GAG ACC CAC CTT GAT CAC CGC GAC ACC ACC GGC CAG CTC ACC GAG GGT ATC GGC TTC GAC AAG GGC TTC GGT GGT GTC GCG GTC ATC AAG GTG GGT GCC GCC CTT GTC GAA CCG GAT AAC CTC GGT GAA CTC CAA ACC GGC CTT GAT GAC AGC AAC CCC GCC GGC CAG 33 33 33 33 33 33 33 33 2.4 General microbial techniques 2.4.1- Media: All media was autoclaved before use. Luria-Bertani (LB): 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% NaCl. LB agar: LB with 1.5% agar. Difco Middlebrook 7H9 broth (BD Biosciences): 4.7g was added to 900ml deionised water and autoclaved. 100ml Middlebrook ADC enrichment (BBL) and 0.05% Tween80 was added before use. Difco Middlebrook 7H10 agar (BD Bioscience): 19g was added to 900ml water and autoclaved. 100ml Middlebrook ADC enrichment and 0.05% Tween80was added before use. 73
Chapter 2 Biofilm base media: 13.6g KH 2 PO 4 , 2g (NH 4 ) 2 SO 4 adjust the pH to 7.2 using KOH pellets then added 0.5mg FeSO 4 .7H 2 O, and 5g Casamino acid. Biofilm media: 94ml biofilm base media with 5ml of 40% glucose, 1ml 0.1M CaCl 2 , 100μl of 1M MgSO 4 . 2.4.2- Additives Additive Stock concentration Working concentration L-Arabinose 20% (w/v) 0.2% (w/v) D-Glucose 20% (w/v) 0.2% (w/v) Ampicillin 100mg/ml 100µg/ml Kanamycin 50mg/ml 50µg/ml Chloramphenicol 50mg/ml 50µg/ml Hygromycin 150mg/ml 150μg/ml IPTG 1 M 1 mM 2.4.3- Growth conditions E. coli growth conditions E. coli was grown at 37°C, unless indicated otherwise, in Luria-Bertani (LB) media in a shaking incubator (180rpm). For overnight cultures, single colonies were picked from a plate or a loop full of frozen culture and inoculated into 5ml of LB. To subculture, 1ml of the 74
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FUNCTIONAL AND STRUCTURAL CHARACTER
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Abstract Proteins are involved in a
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Dedicated to Mum, Dad, and Sharique
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Index of contents Chapter 1: Introd
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2.4.5: Bacteriophage P1 Transductio
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4.1.5: Chimeras DHF & GEI 162 4.2:
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List of illustrations Chapter 1: In
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Fig 4.1: Schematic representation o
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List of tables Chapter 1: Introduct
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SDW STPKs TAE TEMED TBS Sterile dis
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Chapter 1 1.1 Protein Folding The g
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Chapter 1 Fig 1.1: The energy lands
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Chapter 1 To try and escape from th
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Chapter 1 Fig 1.2: Pathways regulat
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Chapter 1 As protein folding in Myc
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Chapter 1 1.4 The molecular chapero
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Chapter 1 1.4.1 Classes of molecula
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Chapter 1 Hsp40 DnaJ CbpA DjlA Ydj1
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Chapter 1 obtained. These revertant
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Chapter 1 1.4.3.1 The ribosome asso
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Page 45 and 46: Chapter 1 1.4.3.4 The small Hsps On
Page 47 and 48: Chapter 1 1.4.3.5 Hsp90 family of c
Page 49 and 50: Chapter 1 Fig 1.11: Structure of Ht
Page 51 and 52: Chapter 1 1.4.3.6 Hsp100 family of
Page 53 and 54: Chapter 1 Fig 1.13: The different r
Page 55 and 56: Chapter 1 encoded by more than one
Page 57 and 58: Chapter 1 Fig 1.15: The illustratio
Page 59 and 60: Chapter 1 (Rommelaere et al., 1993,
Page 61 and 62: Chapter 1 Fig 1.16: Octameric struc
Page 63 and 64: Chapter 1 main difference between t
Page 65 and 66: Chapter 1 1.5.3.3 Prefoldin It has
Page 67 and 68: Chapter 1 result the substrate prot
Page 69 and 70: Chapter 1 Fig 1.18: The structures
Page 71 and 72: Chapter 1 6- If the released peptid
Page 73 and 74: Chapter 1 1.5.4.3 GroEL substrates
Page 75 and 76: Chapter 1 Recent work has suggested
Page 77 and 78: Chapter 1 (A) cpn10 cpn60.1 cpn60.2
Page 79 and 80: Chapter 1 1998). Cpn60.1 however ha
Page 81 and 82: Chapter 1 1.6 Aims of the project T
Page 83 and 84: Chapter 2 Materials and Methods: 2.
Page 85 and 86: Chapter 2 pET-cpn10- cpn60.1 Deriva
Page 87 and 88: Chapter 2 2.3 Primers: Below is a l
Page 89 and 90: Chapter 2 Mut EL.3 Rev groEL 1401-1
Page 91: Chapter 2 29R-EL-60.2 groEL 432-412
Page 95 and 96: Chapter 2 Preparation of bacterioph
Page 97 and 98: Chapter 2 then resuspended in 200µ
Page 99 and 100: Chapter 2 - 10X BSA (depending on t
Page 101 and 102: Chapter 2 - Colony/culture 1μl - S
Page 103 and 104: Chapter 2 The PCR cycling parameter
Page 105 and 106: Chapter 2 tubes were placed on ice
Page 107 and 108: Chapter 2 2.6 Protein analysis 2.6.
Page 109 and 110: Chapter 2 2.6.2- Native gel Reagent
Page 111 and 112: Chapter 2 - Diluted primary antibod
Page 113 and 114: Chapter 2 supernatant was decanted
Page 115 and 116: Chapter 2 2.6.6- Analytical ultrace
Page 117 and 118: Chapter 3 Background As discussed a
Page 119 and 120: Chapter 3 the -35 region of the trp
Page 121 and 122: Chapter 3 20 mins 40 mins 60 mins 8
Page 123 and 124: Chapter 3 (a) 60 mins 90 mins 120 m
Page 125 and 126: Chapter 3 For this experiment, MGM1
Page 127 and 128: Chapter 3 When IPTG was not include
Page 129 and 130: Chapter 3 The results from these co
Page 131 and 132: Chapter 3 3.2.1 The effect of chang
Page 133 and 134: Chapter 3 3.2.2 Using pRARE plasmid
Page 135 and 136: Chapter 3 3.3 Complementation and e
Page 137 and 138: Chapter 3 of MGM100. The use of Tab
Page 139 and 140: Chapter 3 3.3.1 Using pET plasmid i
Page 141 and 142: Chapter 3 1 2 3 4 5 6 7 8 95 kDa 72
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Chapter 3 it only digests the paren
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Chapter 3 72 kDa 1 2 3 4 5 55 kDa 1
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Chapter 3 30°C/26°C 37°C MGM100
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Chapter 3 Fig 3.12: Comparison of t
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Chapter 3 AI90/pACYC-Ptrc-GroESL-Sa
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Chapter 3 1 2 3 4 5 6 7 1000 bp 600
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Chapter 3 10 -1 10 -2 10 -3 10 -4 1
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Chapter 3 this observation. The res
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Chapter 3 expression. However, from
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Chapter 4 Construction and analysis
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Chapter 4 Background In the last ch
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Chapter 4 Experimental design To ma
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Chapter 4 Cpn60.2 7 8 9 Cpn60.1 1 2
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Chapter 4 The full list of all the
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Chapter 4 4.1 Imprecise domain swap
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Chapter 4 4.1.1 Chimera AEC This is
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Chapter 4 (a) Dilutions 10 -1 10 -2
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Chapter 4 of GroEL (fig 4.7). This
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Chapter 4 (a) Dilutions 10 -1 10 -2
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Chapter 4 4.1.5 Chimeras DHF & GEI
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Chapter 4 4.2 Precise domain swaps
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Chapter 4 intermediate domains were
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Chapter 4 1 2 800 kDa 480 kDa 146 k
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Chapter 4 Dilutions 10 -2 10 -3 10
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Chapter 4 is important to note that
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Chapter 4 Summary the complementati
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Chapter 4 The other aspect of this
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Chapter 4 caused due to lack of exp
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Chapter 4 structures were not expec
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Chapter 4 Name Construct Blue -Cpn6
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Chapter 5 Background As discussed i
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Chapter 5 macromolecules at equilib
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Chapter 5 5.1 Purification of JNL 5
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Chapter 5 (a) 1 2 3 4 5 6 7 8 9 10
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Chapter 5 (a) A B C D E (b) 1 2 3 4
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Chapter 5 5.2 AUC analysis of JNL U
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Chapter 5 (a) 1 JNL in Buffer 5 0.9
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Chapter 5 (a) (b) 100 mM P i 75 mM
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Chapter 5 In this experiment GroEL
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Chapter 5 1 2 3 Corrected values Av
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Chapter 5 expected, evidence of lar
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Chapter 5 In summary, based on the
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Chapter 6 In this chapter I will be
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Chapter 6 of Mscpn60.1 can indirect
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Chapter 6 can produce products with
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Chapter 6 As the group in Pittsburg
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Chapter 6 Fig 6.3: Masses of 4 day
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Chapter 6 than 7 days, so there is
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Chapter 6 are required to facilitat
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Chapter 6 Fig 6.4: Sequence alignme
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Chapter 6 To ensure that the lack o
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Chapter 6 6.2.3 Complementation ana
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Chapter 6 Discussion The double rin
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Chapter 6 Section 6.3: Testing the
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Chapter 6 Experimental design For t
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Chapter 6 EcoRV groEL (1.6kb) HindI
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Chapter 6 6.3.2 Complementation res
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Chapter 6 1 2 3 4 72 kDa 55 kDa Fig
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Chapter 6 10 -1 10 -2 10 -3 10 -4 1
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Chapter 6 Discussion The results th
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Summary The main aim of this study
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together, these results strongly su
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Basha, E., K. L. Friedrich & E. Vie
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Cehovin, A., A. R. Coates, Y. Hu, Y
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Diaz-Acosta, A., M. L. Sandoval, L.
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Fux, C. A., J. W. Costerton, P. S.
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Gupta, P., S. Mishra & T. K. Chaudh
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Hoffmann, A., F. Merz, A. Rutkowska
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Ito, K., Y. Akiyama, T. Yura & K. S
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Krzewska, J., G. Konopa & K. Libere
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Lin, Z., D. Madan & H. S. Rye, (200
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Mogk, A. & B. Bukau, (2004) Molecul
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Patzelt, H., G. Kramer, T. Rauch, H
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Rommelaere, H., M. Van Troys, Y. Ga
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Strickland, E., B. H. Qu, L. Millen
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Tyagi, N. K., W. A. Fenton & A. L.
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Yamamori, T. & T. Yura, (1982) Gene
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