Part:BBa_K934001

phaC1-A-B1 [P(3HB) synthesis]

Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.

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.

Fig1.synthesis mechanism of P(3HB)

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.

Fig2.A: E.coli JM109 colonies with BBa_K934001 gene, PHB accumulation. Fig2.B: E.coli JM109 colonies with PlasI-gfp gene, no PHB accumulation.

For more information, see Experience.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 916
  Illegal BglII site found at 1741
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4002