Difference between revisions of "Part:BBa K1149051"

Revision as of 01:51, 1 October 2013

Hybrid promoter phaCAB

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): P(3HB) Purification

The Imperial College iGEM team have successfully purified P(3HB) from E. coli. (MG1655) transformed with either native phaCAB (BBa_K934001 or hybrid promoter phaCAB (BBa_K1149051). Our novel Biobrick hybrid promoter phaCAB (BBa_K1149051) produces significantly 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.]

Comparison of P3HB production P3HB extracted from E.coli MG1655 transformed with left to right, control (empty vector), native phaCAB (BBa_K934001) and hybrid promoter phaCAB (BBa_K1149051). Each produced in 300ml cultures of LB with 3% glucose after one night growing at 37 degrees celsius.

Comparison of P3HB production <b>(left) 1.5ml tube, natural phaCAB (BBa_K934001) (right) 5ml tube, phaCAB expressed from the hybrid promoter, (BBa_K1149051).

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).

J23104+RBS B0034-

Sequence and Features