Composite

Part:BBa_K1149051

Designed by: Margarita Kopniczky   Group: iGEM13_Imperial_College   (2013-09-24)
Revision as of 18:02, 2 October 2013 by Rkelwick (Talk | contribs)

Hybrid promoter phaCAB

Introduction: 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.

Increased production of P(3HB)

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.]

A summary of the improved production of P3HB by our hybrid promoter-phaCAB construct(BBa_K1149051) over the native promoter-phaCAB.
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).


3HB Assay: Determining production of 3HB

|[[File:3HB_assay_from_PHB_másolata.jpg|thumbnail|left|900px| The chemical analysis of the produced bioplastic. The samples break break down to 3HB monomers after treatment with our PhaZ1 enzyme (BBa_K1149010). We synthesised P(3HB) using our improved Biobrick part (hybrid promoter phaCAB, BBa_K1149051). Our engineered bioplastic producing E. coli synthesised P(3HB) directly from waste.

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.

<b>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. Imperial college data

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/Waste_Degradation:_SRF Module 1: Waste to bioplastic]


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+B0034-NATIVE promoter (pwt)+RBS- phaCAB

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 979
    Illegal BglII site found at 1804
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 285
    Illegal NgoMIV site found at 356
    Illegal NgoMIV site found at 956
    Illegal NgoMIV site found at 1268
    Illegal NgoMIV site found at 1547
    Illegal NgoMIV site found at 2199
    Illegal NgoMIV site found at 2221
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4065


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