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 shows the difference between cells storing PHB and those not storing PHB. The cells in blue rectangle area are the cells with PHB synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control.

We cultured the colony in LB solution for 16hrs at 37℃, then we concentrated the solution and painted the letter by the solution on LB agar medium including 0.5μg/ml Nile red and 2% glucose at 37℃ for 36 hours. The cells with PHB would be stained red by Nile red when observed under UV.

FIG2 Difference between cells storing PHB and cells not storing PHB. Blue rectangle: with BBa_K934001 gene, PHB accumulation. Green rectangle: 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