Part:BBa_K1149052

Constitutive phaCAB

This part has a strong E.coli constitutive promoter in front of the phaCAB PHB biosynthetic operon. Nile Red stain, under UV, shown PHB accumulated inside the cells:

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 phaCAB operon, replacing its native promoter. Thus this part consists of an anderson constitutive promoter and the phaCAB operon, which function as an optimised bioplastic producing operon.

Production of P(3HB): Nile Red Staining

O/N cultures of MG1655 transformed with either control (empty vector), native, constitutive or hybrid phaCAB constructs were spread onto LB-agar plates with 3% glucose and Nile red staining.

phaCAB P(3HB) synthesis constructs transformed into MG1655 Strains were grown on Nile red plates, which stain the PHB strongly and fluoresce in presence of PHB. On the left are MG1655 cells with an empty vector (no fluorescence; no plastic), at the bottom is the native promoter (i.e. low fluorescence, some plastic). At the top and right we have our constitutive and hybrid promoter (respectively), which both show high expression and thus fluoresce very clearly.

Conclusion: The red staining indicates the production of P(3HB). More importantly our new Biobricks hybrid promoter phaCAB BBa_K1149051 and constitutive phaCAB BBa_K1149052 produce more P(3HB) than the native phaCAB operon To find more information about the reasons for improvement, the design and methods of changing the promoter on Imperial iGEM wiki: [http://2013.igem.org/Team:Imperial_College/BioPlastic_Recycling:_PHB PHB recycling]

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).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 627
  Illegal BglII site found at 1452
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 604
  Illegal NgoMIV site found at 916
  Illegal NgoMIV site found at 1195
  Illegal NgoMIV site found at 1847
  Illegal NgoMIV site found at 1869
  Illegal NgoMIV site found at 2491
  Illegal NgoMIV site found at 2632
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3713