Part:BBa_K934001
phaC1-A-B1 [P(3HB) synthesis]
The sequence comes from Ralstonia eutropha H16 adapted to become a standard BioBrick part.
Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes synthesized from phaC1，phaA，phaB1，respectively.
The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)
The B1 gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)
The C1 gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)
To synthesize PHB by E.coli, we transformed E.coli JM109 with the constructed phaC1-A-B1 parts on pSB1C3 (BBa_K934001). E.coli JM109 is used to synthesize PHB, because it tends to have a high density accumulation of PHB. As a negative control, we transformed E.coli JM109 with PlasI-gfp on pSB1C3.
The pathway and regulation of Poly[(R)-3-hydroxybutyrate] ,P(3HB) synthesis in Ralstonia eutropha H16 is shown in Fig1. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in phaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in phaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).
Fig2 is the photographs of E.coli colonies on Nile red positive medium taken under UV. The orange colonies in Fig2.A show that the accumulated poly-3-hydroxybutyrate, PHB in cells was stained by Nile red. This result indicates that part BBa_K934001 synthesized PHB. Fig2.B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies.
We cultured the transformant on LB agar medium plates with 0.5μg/ml Nile red and 2% glucose at 37℃ for 30 hours, then we transferred the plates to 4℃ room. After 115 hours, colonies with PHB would be stained Red by Nile red when observed under UV.
For more information, see Experience.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 916
Illegal BglII site found at 1741 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 222
Illegal NgoMIV site found at 293
Illegal NgoMIV site found at 893
Illegal NgoMIV site found at 1205
Illegal NgoMIV site found at 1484
Illegal NgoMIV site found at 2136
Illegal NgoMIV site found at 2158 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 4002
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