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(Ralstonia, Hydrogenovibrio ) (Synechococcus, Spirulina ) (Thiobacillus ) (Rhodobacter, Rhodospirillum ) (Nitrosomonas ) (Chromatium ) (Nitrobacter ) ( Methanobacterium ) Acetyl-CoA (Clostridium ) 3-HP (Chloroflexus ) (3-hydroxypropionate cycle) (Acidianus, Sulfolobus ) TCA (Hydrogenobacter ) (Chlorobium ) (RTCA cycle) (Desulfobacter ) 21
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Image: George Retseck 55
Subsurface lithoautotrophic microbial ecosystems exist in the pores between interlocking mineral grains of many igneous rocks. Autotrophic microbes (green) derive nutrients and energy from inorganic chemicals in their surroundings, and many other microbes (red), in turn, feed on organics created 56
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modified seqence 101
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Clostridium acetobutylicum OXO seqence 105
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TCA 108
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1 T. kodakaraensis KOD1 113
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KOD1 116
KOD1ORF map 117
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KU25 Uracil 120
KW4 pyrf 121
KW4 Southern blot 122
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Rubisco 127
Tk-Rubisco 128
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28 Tk-Rubisco Tk-Rubisco 130
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J Biol Chem. 2002 Aug 30;277(35):31656-62. 136
FOR 137
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FOR 139
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Tk-FOR 142
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HD-1 151
16SrRNAProteobacteria 152
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161
pmg105 pmg103 pmg103 162
PEPCK 163
PEPCK 164
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pm3rbcwt A TN TC PCR B RP TC PCR 168
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pm3rbcwt R. palustris A TN TC PCR B RP TC PCR 170
171 Tk-Rbc Western blot 30 S HS Tk-Rbc SDS-PAGE S: HS:8515 HP: Rb: Tk-Rbc 1, 2 Tk-Rbc
R. palustris Triple mutant pm3rbcwt 172
PCR 10 PCR pm3rbcwt PEPCKpromoter-Tk-rbc DNA Colony No.10 173
PCR PCR Colony No. 1~6 Colony No.7 pm3rbcwt Colony No.8 Triple Mutant RP, TC, TN, 950r : PEPCK promoter, Tk-RbcC, Tk-Rbc N, Tk-Rbc 1kb 174
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PCR pm3rbcwt 1: 2~5: 6~12: YE-acid N: R. palustris Triple mutant (Negative control)p: pm3rbcwt (Positive control)m: λ/hindiii Tk-Rbc Western blot 6~12: 35 6~12 P: Positive 177
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pem7/zeo MCS: 186
PCR pem7/zeo primer 2 3primer 1 3 PCR SalI site 187
pm3rbcwt EM7 promoter PEPCK promoter 188
PCR pm3ezrbcwt, pm3rzrbcwt PCR 189
R. palustris Triple mutant 190
Tk-Rbc Western blot Tk-Rbc (positive control)triple mutant pm3ezrbcwt YE-acid YE-acid 191
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in vitro 193
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Tk-Rubisco R. palustris Rubisco Double mutant DNA Rubisco 195
DNA Rubisco formi-2 1~5 DNA PCR DNA 400ng/l 1l DNA 1, 200ng. 2, 100ng. 3, 50ng. 4, 25ng. 5, 12.5ng Triple mutant DNA 196
Rubisco 197
R. palustris form I-2 Rubisco 198
pem7zeo/tk-rbc BglII, SalI Tk-rbc DNA 199
pwdzrl2tk-rbc 200
PCR Insert Check BamHI 201
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KOD1 Rubisco Rubisco Rubisco Rhodopseudomonas palustris Rubisco Rubisco Rubisco R. palustris Tk-rbc Rubisco Tk-rbc Tk-rbc Tk-rbc Rubisco R. palustris Tk-Rbc TypeIII Rubisco Rubisco Tk-Rubisco Tk-rbc ORF error prone PCR in vitro Tk-Rubisco R. palustris DNA Tk-rbc in vitro, in vivo 206
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RITE FAX 90