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Chapter 2 2.3.2 Primers for site directed mutagenesis Name of Primer Binding site on gene Sequence (5’ to 3’) Length (in bases) ATG-cpn10 For 21 bp upstream of cpn10 to 22 bp into cpn10 AAA TAG TGG AGG GCT CCA ATC ATG GCG AAG GTG AAC ATC AAG C 43 ATG-cpn10 Rev 22 bp in cpn10 to 21 bp upstream of cpn10 GCT TGA TGT TCA CCT TCG CCA TGA TTG GAG CCC TCC ACT ATT T 43 Mut 60.21 For cpn60.2 1332-1359 Mut 60.21 Rev cpn60.2 1359-1332 GGA GGC CCC GCT GGG GCA GAT CGC CTT C GAA GGC GAT CTG CCC CAG CGG GGC CTC C 28 28 Mut 60.22 For cpn60.2 1362-1384 CTC CGG GCT GGC GCC GGG CGT GG 23 Mut 60.22 Rev cpn60.2 1384-1362 CCA CGC CCG GCG CCA GCC CGG AG 23 Mut 60.23 For cpn60.2 1368-1390 GCT GGA GCC GGC CGT GGT GGC CG 23 Mut 60.23 Rev cpn60.2 1390-1368 CGG CCA CCA CGG CCG GCT CCA GC 23 Mut 60.24 For cpn60.2 1371-1393 GGA GCC GGG CGC GGT GGC CGA GA 23 Mut 60.24 Rev cpn60.2 1393-1371 TCT CGG CCA CCG CGC CCG GCT CC 23 Mut EL.1 For groEL 1342-1367 Mut EL.1 Rev groEL 1367-1342 GAA GCT CCG CTG GAA CAG ATC GTA TT AAT ACG ATC TGT TCC AGC GGA GCT TC 26 26 Mut EL.2 For groEL 1371-1393 CTG CGG CGA AGC ACC GTC TGT TG 23 Mut EL.2 Rev groEL 1393-1371 CAA CAG ACG GTG CTT CGC CGC AG 23 Mut EL.3 For groEL 1375-1401 GGC GAA GAA CCG GCT GTT GTT GCT AAC 27 69
Chapter 2 Mut EL.3 Rev groEL 1401-1375 Mut EL.4 For groEL 1378-1404 Mut EL.4 Rev groEL 1404-1378 GTT AGC AAC AAC AGC CGG TTC TTC GCC GAA GAA CCG TCT GCT GTT GCT AAC ACC GGT GTT AGC AAC AGC AGA CGG TTC TTC 27 27 27 Highlighted regions represent specific changes to the original sequences that were used for site directed mutagenesis. 2.3.3 Primers for domain swaps Name of Primer Binding site on gene Sequence (5’ to 3’) Length (in bases) 60.1 flanking fwd 1 cpn60.1 1-25 2F-60.1-60.2 cpn60.1 427-447 3F-60.1-60.2 cpn60.1 1207-1227 4R-60.1-60.2 cpn60.1 429-409 5R-60.1-60.2 cpn60.1 1209-1189 ATG AGC AAG CTG ATC GAA TAC GAC G ACC AAG GAG CAG ATC GCG CAG GTG GCG ACG GTG CGC AAT GCC AAG GCC GCG GTC GAG GAG GGC ATC TGC GGT GGC CGC GAT GCC GGT CTT GCC GGA CAC CTC CTC GAC GGC GGC CTT GGC GGC CGC GAC CGC 25 33 33 33 33 60.1 flanking rev 6 cpn60.1 1620-1600 TCA GTG CGC GTG CCC GTG GTG 21 60.2 flanking fwd 7 cpn60.2 1-25 8F-60.2-60.1 cpn60.2 427-447 9F-60.2-60.1 cpn60.2 1207-1227 10R-60.2-60.1 cpn60.2 429-409 ATG GCC AAG ACA ATT GCG TAC GAC G GGC AAG ACC GGC ATC GCG GCC ACC GCA GCG ATT GCG GCC GCC AAG GCC GCC GTC GAG GAG GGC ATC CGC CAC CTG CGC AAT CTG CTC CTT GGT CTC GAC 25 33 33 33 70
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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
Page 37 and 38: Chapter 1 obtained. These revertant
Page 39 and 40: Chapter 1 1.4.3.1 The ribosome asso
Page 41 and 42: Chapter 1 (A) (B) Fig 1.7: Structur
Page 43 and 44: Chapter 1 complete the reaction cyc
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: Chapter 2 2.3 Primers: Below is a l
Page 91 and 92: Chapter 2 29R-EL-60.2 groEL 432-412
Page 93 and 94: Chapter 2 Biofilm base media: 13.6g
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
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Chapter 3 3.3.1 Using pET plasmid i
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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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