We used E. coli that expresses PhaCAB enzymes to make P3HB bioplastic. This is tested on nile red plates which stains P3HB.

E.coli transformed with the phaCAB operon(native, hybrid and constitutive are different promoters expressing the operon) fluoresce when grown on LB-Agar plates with Nile Red stain. The P3HB produced within them is stained. Those without the operon do not(EV- transformed with empty pSB1C3). When the promoter is changed from the native form to the hybrid or constitutive J23104 then the fluorescence is more intense, indicating increased P3HB production.

Using model simulation results, we predict that the concentration of PhaCAB enzymes

Since our model predicts that the concentration of PhaB enzymes is the rate limiting step in P3HB production, we designed a hybrid promoter consists of the J23104 constitutive promoter and the native promoter to optimise gene expression. Our results show that we have successfully produced 10-fold more P3HB bioplastic compared with the native promoter.

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 . (left) 1.5ml tube, natural phaCAB (BBa_K934001) (right) 5ml tube, phaCAB expressed from the hybrid promoter, (BBa_K1149051).

One of the objectives of Module 1 is to produce P3HB bioplastic from waste. By comparing degradation product 3HB of P3HB bought from Sigma, produced from glucose and produced from the waste, we found there is no significant differences in 3HB concentration between these samples.

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. Imperial iGEM data