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Chapter 2 2.2 Plasmids The pTrc expression plasmids used in this project were constructed by Dr. Yanmin Hu (St George’s Hospital, London), and derived from the expression plasmid ptrc99A (fig 2.1). This plasmid has an amp R selective marker, and contains a multiple cloning site downstream from an IPTG inducible Ptrc promoter. Plasmid Features/Genes Expressed Reference pTrc99A pTrc-groESgroEL pTrc-groEScpn60.1 pTrc-groEScpn60.2 pTrc-cpn10- cpn60.1 An expression plasmid driven by the trc promoter (-35 region of the trp promoter together with the -10 region of the lac promoter), inducible by IPTG. A derivative of the pTrc plasmid containing E.coli groEL and groES genes under the control of the Ptrc promoter. A derivative of the pTrc plasmid containing E.coli groES gene along with M. tuberculosis cpn60.1 gene under the control of the Ptrc promoter. A derivative of the pTrc plasmid containing E.coli groES gene along with M. tuberculosis cpn60.2 gene under the control of the Ptrc promoter. A derivative of the pTrc plasmid containing M. tuberculosis cpn10 gene and the cpn60.1 gene under the control of the Ptrc promoter. Amann et al., 1988 Lund, Unpublished Lund, Unpublished Lund, Unpublished Lund, Unpublished pTrc-cpn10- cpn60.2 A derivative of the pTrc plasmid containing M. tuberculosis cpn10 gene and the cpn60.2 gene under the control of the Ptrc promoter. Lund, Unpublished pTrc-cpn60.2 A derivative of the pTrc plasmid expressing M. tuberculosis Cpn60.2 This study pBAD24 Expression plasmid driven by arabinose inducible P BAD promoter Guzman et al., 1995 pRARE Plasmid encoding tRNAs for codons that rarely occur in E. coli Novagen pET23a+ Expression plasmid driven by the T7 promoter Novagen pET-groEScpn60.1 Derivative of pET23a+ expressing E. coli GroES and M. tuberculosis Cpn60.1 This study 65
Chapter 2 pET-cpn10- cpn60.1 Derivative of pET23a+ expressing M. tuberculosis Cpn10 and Cpn60.1 This study pSMGroEL1- H12 Expression plasmid containing M. smegmatis Cpn10 and Cpn60.1 Ojha et al., 2005 pMs10- mtbcpn60.1 pSMGroEL1-H12 derivative expressing M. smegmatis Cpn10 and M. tuberculosis Cpn60.1 This study 2.2.1 Plasmid maps: Plasmid map of pTrc99a Fig 2.1: Plasmid map of pTrc99a. Image obtained using Plasmapper (Dong et al., 2004) 66
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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
Page 33 and 34: Chapter 1 1.4.1 Classes of molecula
Page 35 and 36: 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: Chapter 2 Materials and Methods: 2.
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 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
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Chapter 3 3.3 Complementation and e
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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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