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Chapter 3 3.5 Random mutagenesis of Cpn60.1 It is already know that in E. coli a relatively small concentration of GroEL is needed to sustain cell growth at 37°C (Herendeen et al., 1979, VanBogelen et al., 1987, Lorimer, 1996). It was discussed in section 3.1 that even after 3 hours of being induced with arabinose, GroEL bands could not be distinguished on the SDS-PAGE gel without using western blots. Although attempts to improve the overall level of expression of Cpn60.1 in the last few experiments were unsuccessful, it can still be speculated that, if Cpn60.1 could function as a replacement for GroEL in E. coli then the level of expression seen in MGM100 with GroES should have been sufficient to do so. However, it is unknown how active Cpn60.1 is compared with GroEL, so it is possible that if its activity is much lower than GroEL then a higher concentration of Cpn60.1 would be needed to complement. All the experiments done so far on Cpn60.1 have shown that it cannot complement in E. coli in the absence of endogenous GroEL, while Cpn60.2 is very capable of sustaining cell growth up to 37°C. Cpn60.1 and Cpn60.2 share 61% sequence identity and 76% sequence similarity (Fig 3.12). It was hypothesised that it may be possible to obtain a functional form of Cpn60.1 that could complement for the loss of endogenous GroEL in E. coli MGM100 with random mutagenesis. For this reason, the XL1-Red E. coli strain provided by Agilent technologies (formerly Stratagene) was used to mutagenise cpn60.1. This E. coli strain is deficient in three of the primary DNA repair pathways and according to its manufacturer, has a mutation rate ~5000 fold higher than that of wild-type. Unfortunately, after several attempts, no such mutants were found. 129
Chapter 3 Fig 3.12: Comparison of the amino acid sequence of M. tuberculosis chaperonins. The asterisk indicates identical residues, a colon indicates a conserved substitution, and a full stop indicates a semi conserved substitution. The sequence was aligned with the help of ClustalW2 on the European Bioinformatics Institute website. 130
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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
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Chapter 1 6- If the released peptid
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Chapter 1 1.5.4.3 GroEL substrates
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Chapter 1 Recent work has suggested
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Chapter 1 (A) cpn10 cpn60.1 cpn60.2
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Chapter 1 1998). Cpn60.1 however ha
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Chapter 1 1.6 Aims of the project T
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Chapter 2 Materials and Methods: 2.
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Chapter 2 pET-cpn10- cpn60.1 Deriva
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Chapter 2 2.3 Primers: Below is a l
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Chapter 2 Mut EL.3 Rev groEL 1401-1
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Chapter 2 29R-EL-60.2 groEL 432-412
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Chapter 2 Biofilm base media: 13.6g
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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
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: Chapter 3 30°C/26°C 37°C MGM100
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
Page 173 and 174: Chapter 4 4.1.1 Chimera AEC This is
Page 175 and 176: Chapter 4 (a) Dilutions 10 -1 10 -2
Page 177 and 178: Chapter 4 of GroEL (fig 4.7). This
Page 179 and 180: Chapter 4 (a) Dilutions 10 -1 10 -2
Page 181 and 182: Chapter 4 4.1.5 Chimeras DHF & GEI
Page 183 and 184: Chapter 4 4.2 Precise domain swaps
Page 185 and 186: Chapter 4 intermediate domains were
Page 187 and 188: Chapter 4 1 2 800 kDa 480 kDa 146 k
Page 189 and 190: Chapter 4 Dilutions 10 -2 10 -3 10
Page 191 and 192: Chapter 4 is important to note that
Page 193 and 194: Chapter 4 Summary the complementati
Page 195 and 196: Chapter 4 The other aspect of this
Page 197 and 198: 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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