PHB production

During our project we successfully synthesised the bioplastic P(3HB). We have stained it in the cells on the agar plate and are working hard to do so under the microscope as well. We succesfully extracted PHB from cultures and would also like to detect 3HB monomers from the "white stuff" as an elegant proof of chemical composition.

Nile red staining

O/N cultures of MG1655 transformed with either control or phaCAB plasmid were spread onto LB-agar plates with 3% glucose and Nile red staining.

Initial work with plastic synthesis in the native promoter. Nile red staining was used to show expression of the plastic by fluorescence imaging. Control cells with empty vector are shown on the left, while native phaCAB transformed MG1655 is on the right.

MG1655 constructs synthesising plastic. 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 [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1149051 hybrid promoter phaCAB BBa_K1149051] and [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1149052 constitutive phaCAB BBa_K1149052] produce more P(3HB) than the native phaCAB operon

Extraction of P3HB

We extract P3HB using a technique which first disrupts the cell membranes and then degrades the remaining parts of the cell with bleach. See the protocols section for more details.

Once the cultures have been centrifuged and the supernatant poured off , the biomass is clearly seen as cell pellets.

Cell pellets left after the centrifugation of 300ml LB media with 3% glucose.

Cell pellets left after the centrifugation of 300ml LB media with 3% glucose. These pellets have slid back into the remaining supernatant.

P(3HB) extracted from phaCAB transformed MG1655 that were grown in LB with 3% glucose

P(3HB) extracted from phaCAB transformed MG1655 that were grown in LB with 3% glucose

P(3HB) extracted from phaCAB transformed MG1655 that were grown in M9M mixed waste.

Comparison of P3HB production P3HB extracted from E.coli MG1655 with pSB1C3 containing, from left to right, nothing, natural phaCAB(BBa_K934001) and phaCAB expressed from the hybrid promoter, (BBa_K1149051). Each produced in 300ml cultures of LB with 3% glucose after one night growing at 37 degrees celsius.

3HB assay

We are proving that the white, occasionally gray, stuff that we got after the extraction process is actually really certainly Poly-3-Hydroxy-Butyrate. And that it can be degraded by phaz1.

We are hydroxybutyrate colorimetric assay kit. You can download the description of the product and the protocol here. The chemical reaction behind the assay is illustrated on the image below: