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Chapter 3 Since a GroEL band wasn’t distinguishable on the Coomassie stained gel in either arabinose or glucose, its presence was determined with western blotting (Fig 3.2). The results of the western blot confirmed the presence of GroEL in the samples from cultures grown with arabinose as well as very low levels in the samples from cells grown with glucose. This was expected, and also confirms that the P BAD promoter can be used to express or repress GroEL in MGM100. This also demonstrated that even after 1.5 hours (approximately 4-5 generations), although the levels of GroEL in the glucose culture was extremely low, there was still enough to sustain some growth. This result was again not surprising as it has been shown by previous work conducted in the lab that a very small concentration of GroEL is required to keep the cells viable (Ivic et al., 1997, Lorimer, 1996). It was however noted that after the initial 3 hour incubation period, cell growth in glucose drastically reduced when compared with those in arabinose. Cells grown on arabinose show a much more prominent GroEL band on the film. It was observed that the expression level of MetE started to rise at about 2 hours after incubation in the presence of glucose and not in the presence of arabinose. Taken together, these results confirmed the earlier findings that the reduction in the levels of GroEL within E. coli causes over-expression of the newly translated MetE protein. This result can also be used as a potential visual marker for a lack of functional chaperonin in a cell. 103
Chapter 3 (a) 60 mins 90 mins 120 mins 180 mins 1 2 3 4 5 6 7 8 9 95 kDa 72 kDa 55 kDa 60 mins 90 mins 120 mins 180 mins (b) 1 2 3 4 5 6 7 8 Fig 3.2: Western blot of GroEL from MGM100 grown in arabinose or glucose. (a) SDS- PAGE protein gel of MGM100 grown in arabinose or glucose (arrows pointing at MetE). (b) Western blot using GroEL specific antibody. In both cases odd numbered lanes are MGM100 in arabinose, while even numbered lanes are MGM100 in glucose. Lane 9- Prestained protein marker (Fermentas). 104
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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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Chapter 1 (A) (B) Fig 1.7: Structur
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Chapter 1 complete the reaction cyc
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Chapter 1 1.4.3.4 The small Hsps On
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Chapter 1 1.4.3.5 Hsp90 family of c
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Chapter 1 Fig 1.11: Structure of Ht
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Chapter 1 1.4.3.6 Hsp100 family of
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Chapter 1 Fig 1.13: The different r
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Chapter 1 encoded by more than one
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Chapter 1 Fig 1.15: The illustratio
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Chapter 1 (Rommelaere et al., 1993,
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Chapter 1 Fig 1.16: Octameric struc
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Chapter 1 main difference between t
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Chapter 1 1.5.3.3 Prefoldin It has
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Chapter 1 result the substrate prot
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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 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: Chapter 3 20 mins 40 mins 60 mins 8
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
Page 143 and 144: Chapter 3 it only digests the paren
Page 145 and 146: Chapter 3 72 kDa 1 2 3 4 5 55 kDa 1
Page 147 and 148: Chapter 3 30°C/26°C 37°C MGM100
Page 149 and 150: Chapter 3 Fig 3.12: Comparison of t
Page 151 and 152: Chapter 3 AI90/pACYC-Ptrc-GroESL-Sa
Page 153 and 154: Chapter 3 1 2 3 4 5 6 7 1000 bp 600
Page 155 and 156: Chapter 3 10 -1 10 -2 10 -3 10 -4 1
Page 157 and 158: Chapter 3 this observation. The res
Page 159 and 160: Chapter 3 expression. However, from
Page 161 and 162: Chapter 4 Construction and analysis
Page 163 and 164: Chapter 4 Background In the last ch
Page 165 and 166: Chapter 4 Experimental design To ma
Page 167 and 168: Chapter 4 Cpn60.2 7 8 9 Cpn60.1 1 2
Page 169 and 170: Chapter 4 The full list of all the
Page 171 and 172: 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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