Difference between revisions of "Part:BBa K1149051"
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<partinfo>BBa_K1149051 short</partinfo> | <partinfo>BBa_K1149051 short</partinfo> | ||
+ | <h2>Introduction: Optimised bioplastic producing operon</h2> | ||
In R. eutropha cells, P(3HB) is made through 3 steps. Two acetyl-CoA molecules made from carbohydrate converted to acetoacetyl-CoA by acetyl-CoA acetyltransferase encoded by PhaA gene. Then acetoacetyl-CoA reductase encoded by PhaB gene catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA (3HB-CoA). Finally, P(3HB) synthase encoded by PhaC gene catalyzes polymerization reaction of monomer molecules to polymer P(3HB) [2]. | In R. eutropha cells, P(3HB) is made through 3 steps. Two acetyl-CoA molecules made from carbohydrate converted to acetoacetyl-CoA by acetyl-CoA acetyltransferase encoded by PhaA gene. Then acetoacetyl-CoA reductase encoded by PhaB gene catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA (3HB-CoA). Finally, P(3HB) synthase encoded by PhaC gene catalyzes polymerization reaction of monomer molecules to polymer P(3HB) [2]. | ||
− | We cloned the constitutive promoter J23104+RBS B0034 upstream of the native phaCAB promoter and the phaCAB operon. Thus this part | + | We cloned the constitutive promoter J23104+RBS B0034 upstream of the native phaCAB promoter and the phaCAB operon. Thus this part consists of a hybrid promoter system, which functions as an optimised bioplastic producing operon. |
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+ | <h2>Production of P(3HB): Nile Red Staining</h2> | ||
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+ | <h2>Production of P(3HB): P(3HB) Purification</h2> | ||
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+ | <h2>References</h2> | ||
+ | References | ||
1. H.Ohara. Change from Oil-based to Bio-based. Sen’i gakkaishi 66, 4.129-132 (2010) | 1. H.Ohara. Change from Oil-based to Bio-based. Sen’i gakkaishi 66, 4.129-132 (2010) | ||
2. Pohlmann, A. et al. Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nature biotechnology 24, 1257–62 (2006). | 2. Pohlmann, A. et al. Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nature biotechnology 24, 1257–62 (2006). | ||
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Revision as of 22:52, 30 September 2013
Hybrid promoter phaCAB
Introduction: Optimised bioplastic producing operon
In R. eutropha cells, P(3HB) is made through 3 steps. Two acetyl-CoA molecules made from carbohydrate converted to acetoacetyl-CoA by acetyl-CoA acetyltransferase encoded by PhaA gene. Then acetoacetyl-CoA reductase encoded by PhaB gene catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA (3HB-CoA). Finally, P(3HB) synthase encoded by PhaC gene catalyzes polymerization reaction of monomer molecules to polymer P(3HB) [2].
We cloned the constitutive promoter J23104+RBS B0034 upstream of the native phaCAB promoter and the phaCAB operon. Thus this part consists of a hybrid promoter system, which functions as an optimised bioplastic producing operon.
Production of P(3HB): Nile Red Staining
Production of P(3HB): P(3HB) Purification
References
References 1. H.Ohara. Change from Oil-based to Bio-based. Sen’i gakkaishi 66, 4.129-132 (2010) 2. Pohlmann, A. et al. Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nature biotechnology 24, 1257–62 (2006).
J23104+RBS B0034-
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 979
Illegal BglII site found at 1804 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 285
Illegal NgoMIV site found at 356
Illegal NgoMIV site found at 956
Illegal NgoMIV site found at 1268
Illegal NgoMIV site found at 1547
Illegal NgoMIV site found at 2199
Illegal NgoMIV site found at 2221 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 4065