Team:Imperial College/mainresults

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Main Results

  • Resource-full Waste
  • Plastic Fantastic
We made P3HB bioplastic

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.


Modelling

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


We optimised bioplastic production

Since our model predicts that the concentration of PhaB enzymes is the rate limiting step in P3HB production, we designed a [http://parts.igem.org/Part:BBa_K1149051 hybrid promoter] consists of the J23104 constitutive promoter and the native promoter to optimise gene expression. Our results show that we have successfully produced 11-fold more P3HB bioplastic compared with the native promoter.

Comparison of P3HB production . (left) 1.5ml tube, natural phaCAB (BBa_K934001) (right) 5ml tube, phaCAB expressed from the hybrid promoter, (BBa_K1149051).
A summary of the improved production of P3HB by our hybrid promoter-phaCAB construct(BBa_K1149051) over the native promoter-phaCAB.


We made bioplastic from mixed waste

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

3HB_from_PHB_from_waste.jpg

E. coli can feed on 3HB


We degraded P3HB

Our clearing zone assay indicates that P3HB depolymerase PhaZ1 started to fairly quickly degrade P3HB from the first day. After 3 days, there is evidently a clear zone around the well containing PhaZ1. We are the first iGEM team to degrade bioplastics!

PhaZ1 showing P3HB degradation ability by generating a clear zone on a P3HB LB agar plate. A. No difference between empty vector cell lysate and PhaZ1 cell lysate right after they were pipetted into the wells. B. PhaZ1 in the cell lysate started to clear P3HB around the well after 1 day. C. PhaZ1 created a clear zone around the well after 3 days, whereas it was still cloudy around the empty vector cell lysate well.

We degraded P3HB we made from waste

Using 3HB colourimetric assay kit, we have shown that we have degraded the P3HB made from waste into 3HB monomers. In addition, there is no significant difference in 3HB concentration between different P3HB sources. This result proves that we now have a closed loop for P3HB bioplastic recycling!


We degraded PLA

Content

Our Sponsors

TueSponsorsEppendorf.png 125px Invitrogen.jpg Geneart.jpg CSynBI.JPG